P53 Tumor Suppressor Gene and Herbs

p53 Tumour Suppressor Gene
Cells are incessantly bombarded by an assortment of environmental and intrinsic factors that cause cellular damage. Although mild damage is often reparable, extensive damage poses a potential oncogenic danger. In the latter case, the benefit of the organism calls for the eradication of the potentially life-threatening cells, which often is achieved through activation of an apoptotic cell death program. Thus, the cell is continually faced with an agonizing choice: repair and live, or die (Aylon & Oren 2007).

Mutations of p53, a tumor suppressor gene, are known to be involved in the pathogenesis of a number of neoplasms. Findings support the hypothesis that p53 mutations are homogeneous throughout a tumor and may thus be a more useful diagnostic and prognostic indicator than the expression of p53, which does not reliably correlate with p53 mutations.
P53 has many anticancer mechanisms, and plays a role in apoptosis, genetic stability, and inhibition of angiogenesis. In its anti-cancer role, p53 works through several mechanisms:
1. It can activate DNA repair proteins when DNA has sustained damage.
2. It can induce growth arrest by holding the cell cycle at the G1/S regulation point on DNA damage recognition (if it holds the cell here for long enough, the DNA repair proteins will have time to fix the damage and the cell will be allowed to continue the cell cycle).
3. It can initiate apoptosis, the programmed cell death, if DNA damage proves to be irreparable.
Tumour-suppressor genes are regulators that control the expression of other genes. Normal cells contain dominant suppressor genes that down regulate cancer genes.
Mutations that knock out tumour-suppressor genes are far more threatening than the activation of oncogenes. The genetic most common mutation is in tumour-suppressor p53. p53 is a repair gene. 70% of all colon cancers, 30 to 50% of breast and ovarian, 50% of all lung cancers, all small-cell lung cancers and most prostate cancers are a result of a defect in p53 gene.
In response to mild reparable damage, the tumour suppressor protein p53 is believed to trigger transient cell cycle arrest allowing sufficient time for repair of the damage and re-entry into a normal cell cycle. In contrast, severe, extended or irreparable DNA damage will often lead to apoptosis. These different fates are largely orchestrated through the differential activation of distinct subsets of p53 target genes.
The p53 tumour suppressor protein acts as a major Defense against cancer. Among its most distinctive features is the ability to elicit both apoptotic death and cell cycle arrest. In the journal Cell, Das et al. (2007) and Tanaka et al. (2007) provide new insights into the mechanisms that dictate the life and death decisions of p53.
The cell is continually faced with an agonizing choice: repair and live, or die. Defects in this decision process can lead to cancer, and insights into the mechanisms of dysregulation can improve strategies for designing more effective therapies. This cell fate choice often depends on p53. Sitting at the junction of an extremely complex network of cellular signalling, p53 assimilates disparate input signals such as oncogene activation, DNA damage, mitotic impairment or oxidative stress to initiate appropriate outputs—DNA repair, cell cycle arrest, senescence, or apoptosis (Harris and Levine, 2005).
Approximately half of all cancers bear p53 gene mutations, the vast majority of which impair the ability of p53 to act as a sequence-specific transcriptional activator (Cell 2007).
p53 is a tumor suppressor protein that regulates the expression of a wide variety of genes involved in Apoptosis, Growth arrest, Inhibition of cell cycle progression, Differentiation and accelerated DNA repair or Senescence in response to Genotoxic or Cellular Stress. As a transcription factor, p53 is composed of an N-terminal Activation Domain, a central specific DNA Binding Domain, and a C-terminal Tetramerization Domain, followed by a Regulatory Domain rich in basic Amino acids. Having a short half-life, p53 is normally maintained at low levels in unstressed mammalian cells by continuous ubiquitylation and subsequent degradation by the 26S Proteasome. Nonphosphorylated p53 is ubiquitylated by the MDM2 (Mouse Double Minute-2) ubiquitin ligase. MDM2 binding inactivates p53 by two mechanisms.
First, MDM2 binds to the transactivation domain of p53, precluding interaction with the transcriptional machinery. Second, this binding mediates the covalent attachment of ubiquitin to p53. Ubiquitylated p53 is then degraded by the Proteasome.
Thus MDM2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. When the cell is confronted with stress like DNA damage, Hypoxia, Cytokines, Metabolic changes, Viral infection, or Oncogenes, however, p53 ubiquitylation is suppressed and p53 accumulates in the nucleus, where it is activated and stabilized by undergoing multiple covalent modifications including Phosphorylation and Acetylation.
Sulforaphane, Phenethyl isothiocyanate from cruciferous vegetables, Proanthocyanidins from berries, beans and wine, Carotenoids from peas, cabbage, tomato, orange, carrots, pumpkin and spinach, Terpenoids from citrus, cherries, grapes, Polyphenols, including Curcumin, Resveratrol, and 6-Gingerol, also Indole-3-carbinol (I3C) all regulate p53 expression (Aravindaram & Yang 2010).
The protein expression of p53, p21 affected by emodin (from Rheum emodi, a Himalayan rhubarb) were determined by Western blot, and Fas by immunocytochemistry.
Results: Emodin significantly inhibited cell growth of Human Hepatoma cell line smmc-7721 and the molecular mechanism of apoptosis may be p53-dependent, furthermore Fas may also play an important role in the apootosis (Fan et al 2008).
Salvianolic acid A is one of the active components from Salvia miltiorrhiza, which was found to suppress the growth of mouse tumors. The mechanisms may be the suppression of the over-expression of c-myconcogene, the inhibit the function of Ras oncoprotein, the increase the expression of P53 tumor suppressor gene and the interrupt P46-associated mitogen-activated pathway other than farnesylation of Ras protein (Li et al 2002).
Conjugated Linoleic Acid (CLA) inhibits the proliferation of human breast cancer cells (MCF-7), induced by estradiol and insulin (but not EGF). In fact, CLA caused cell kill (cytotoxicity) when tumour cells were induced with insulin (Chujo et al. 2003). The antiproliferative effects of CLA are partly due to their ability to elicit a p53 response that leads to growth arrest (Kemp et al. 2003). CLA elicits cell killing effects in human breast tumour cells through both p53-dependent and p53 independent pathways according to the cell type (Majumder et al. 2002).
The inhibitory effect on growth of melanoma in vivo were examined by mice melanoma models transplanted B16 cells to C57BL/6J mice. MTT method was used to assay the contribution of curcumin to B16 cells in vitro. The apoptosis and expression of Bcl-2 ,P53 gene of B16 cells were analyzed by flow cytometry, and HPLC assay was used to detect the change of GSH in B16 melanoma tissues. The growth inhibitory effect of curcumin on mouse melanoma is proved in vivo and in vitro respectively. Curcumin can induce some ceils to apoptosis which may be relevant to down-regulation of bcl-2 expression and up-regulation of p53 expression (Gui et al 2008).
Exposure of human prostate cancer cells (PC-3, LNCaP, and C4-2) to honokiol resulted in apoptotic DNA fragmentation in a concentration- and time-dependent manner irrespective of their androgen responsiveness or p53 status. Honokiol-induced apoptosis correlated with induction of Bax, Bak, and Bad and a decrease in Bcl-xL and Mcl-1 protein levels. Transient transfection of PC-3 cells with Bak- and Bax-targeted siRNAs and Bcl-xL plasmid conferred partial yet significant protection against honokiol-induced apoptosis. Oral gavage of 2 mg honokiol/mouse (thrice a week) significantly retarded growth of PC-3 xenografts without causing weight loss. Tumors from honokiol-treated mice exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors (Hahm  et al 2008).

Herbs induce apoptosis via tumor suppressor gene p53
ImmunohistochemicaI staining tests revealed that the expression of Bcl-2 protein was down-regulated, and that of P53 protein was up-regulated after Agrimonia Pilosa water extract, which could inhibit the proliferation of human esophageal cancer cell llne Eta109 cells in vitro by down-regulating the expression of Bcl-2 and up-regulating the expression of P53 protein (Ma et al 2007).
The Chinese herbal mixture, Antitumor B (ATB), also known as Zeng Sheng Ping, is a botanical agent composed of six Chinese herbs: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus, and Dioscorea bulbifera. As p53 and p16 are among the most common alterations observed in human lung cancer, defects in the tumor suppressor gene p53- and p16-dependent pathways may be a significant impediment to successful cancer prevention. Results suggest that ATB is an effective chemopreventive against mouse lung tumorigenesis. Furthermore, ATB exhibited an enhanced inhibitory effect in animals harboring genetic alterations (Zhang et al 2004).
Bupleurum falcatum Saikosaponin D, one of the major components of Bupleurum falcatum which could be extracted from other species of Bupleurum and from related genuses, is used for the treatment of various liver diseases in traditional Chinese medicine(14). It can inhibit the proliferation in the lung cancer cell line, A549, with the IC50 value at 10.18 + 0.09 M. By using flow cytometry and PI staining, Saikosaonin D 10 M increased the population of cells in the G1 phase from 34.7% to 53.9%.
Marked induction of p53 and p21/WAF1 protein was observed in a dose-dependent manner, indicating that the Saikosaponin D-mediated cell cycle arrest might operate through the induction of p21/WAF1 protein on a p53-dependent event in A549 cells. The caspase 8 activity increased at 12 hours and reached maximum induction at 24 hours in 20 M Saikosaponin D treated A549 cells (Hsu et al 2004 a).
Curcuma longa
Curcumin, a phenolic compound of the rhizome of the plant Curcuma longa has anti-inflammatory, antioxidant and anticancer activities. A549 and H1299 human lung cancer cell lines were used for the present study and found that the growth inhibitory effect of curcumin was concentration dependent in both cell lines. The IC50s at 24 hours exposure of curcumin were 50 and 40 M in A549 and H1299 cells, respectively. Determination of PARP cleavage in curcumin treated H1299 cells by flow cytometry, at 24 hours, a concentration at 100 M of curcumin induced PARP cleavage in approximately 90% of treated cells.
Induction of apoptosis by curcumin involved several pathways. RT-PCR was performed and found that a decrease in expression of p53, bcl-2 and bcl-XL was observed. Bax and caspase genes remained unchanged up to the 60 M of curcumin but showed a decrease in expression levels at 80-160 M (Pillai  et al 2004).
Scutellaria barbata, a traditional Chinese herbal medicine native to southern China, is widely used as an anti-inflammatory and a diuretic in China. Extracts of Scutellaria barbata have been shown to have in vivo growth inhibitory effects on a number of cancers such as S180 mouse sarcoma, U14 cervical carcinoma, solid hepatoma, etc. The extract of Scutellaria barbata on human lung cancer cells, A549, exhibited a marked growth inhibitory effect in a dose-dependent manner. The IC50 was approximately 0.21 + 0.04 mg/ml.
Apoptosis of A549 cells induced by Scutellaria barbata was analyzed by Annexin-V staining and demonstrated that treated cells with 0.5 mg/ml Scutellaria barbata extract for 48 hours resulted in a rate of cell apoptosis of 57.67%. The cDNA microarray experiment was performed to characterize the mechanism of Scutellaria barbata induced killing and found that two genes related to cell response to DNA damage, GADD45A and GIP, decreased dramatically. A total of 20 cell cycle genes were found to have changed after treatment indicated that the cell cycle was widely involved in the Scutellaria barbata treatment. Some enzyme activity (STK12,DUSP5 and TOPK), cell signal transduction (GIP, BMP2), nucleic acid binding (ATF3,HNRPD and SMARCF1)were also found to be affected (Yin et al 2004).
Herba Scutellaria barbatae, was cytotoxic to 100% (11 of 11) of actively proliferating ovarian lines tested and 50% (2 of 4) of actively proliferating breast cell lines tested. Confluent cultures were resistant to killing by herb, whereas subconfluent cultures were sensitive. Resistant proliferating cell lines expressed higher levels of bcl2 (Powell et al 2003).
Flavonoid compounds
Flavonoids are a broadly distributed class of plant pigments, universally present in vascular plants and responsible for much of the coloring in nature. They are strong antioxidants that occur naturally in foods and can inhibit carcinogenesis in rodents.
Acacetin, a flavonoid compound found in Robinia pseudoacacia, has been reported to possess anti-peroxidative, anti-inflammatory and antiplasmodial effects (Kraft et al 2003). The proliferation inhibitory effect of acacetin was observed to be in a dose dependent manner with A549 human lung cancer cell lines. The effect of acacetin on cell cycle progression of A549 with 5 μM increased the population of the G1 phase from 34.7 to 42.6%. DNA fragmentation of A549 was found at 12 hours and maximized at 48 hours after exposure of A549 cells to acacetin with 5 and 10 μM. The level of p53, p21, Fas and Fas ligand were assayed by using the ELISA kit and found that acacetin increased the expression of p53 and p21/WAF1 proteins in A549 cells(29). Results on the Fas ligand assay indicated that FasL, mFasL and sFasL increased in a dose-dependent manner.
Isoliquiritigenin (ISL), a flavonoid found in licorice and shallot, is a potent anti-oxidant with antiinflammatory, antiplatelet aggregation and cancer preventing properties. The ISL inhibited the proliferation of A549 cells with IC50 value at 27.14 mol/L. Compared with the control group, 20 mol/L ISL increased the population of cells in the G1 phase from 26.3 to 45.8%. DNA fragmentation was found after the addition of ISL at the level of 20 and 40 mol/L at 12 hours and maximal at 48 hours. The number of cells undergoing apoptosis at 48 hours increased approximately 3.2 fold with ISL 20 mol/L and 7.1 fold with 40 μmol/L. The expression of p53 and p21/WAF1 protein was assayed by ELISA and showed a marked induction of p53 and p21/WAF1 protein with a maximum level at 12 and 24 hours respectively. Maximum Fas was detected at 24 hours with a similar result as Fas ligand (Hsu et al 2004 b).
Natural stilbenoids, including resveratrol which is originally identified as a phytoalexin abundant in grapes, peanuts, pines and other Leguminosae family plants, have been reported to exhibit a variety of important biological effects such as a protective role in atherosclerosis and coronary heart diseases. Many studies have also demonstrated the potential of natural stilbenoids to mediate the strong antioxidant, anti-mutagenic, anti-inflammatory or potent cancer chemopreventive effects in carcinogenesis (Jang et al 1997). In addition, resveratrol inhibited the growth of several human cancer cell lines, including human oral squamous carcinoma, promyelocytic leukemia, breast, prostate and colon cancer cells (Schneider et al 2000). The growth inhibitory potential of stilbenoids was determined in cultured A549 cells; one of these stilbenoids, 3, 4, 5- trimethoxy-4’-bromo-sis-stilbene (BCS) exhibited a remarkable growth inhibitory effect against A549 lung cancer cells with a IC50 value 0.03 M. When treated cells were analyzed for the cell cycle; the G2/M phase was accumulated in a time-dependent manner of up to 16 hours and then subsequently increased in the sub- G1 phase, indicative of apoptotic peaks during incubation time. Apoptosis of treated cells was confirmed by the DNA ladder pattern. Western blotting was performed and it was found that the p53 protein level was increased after 8 hours incubation with BCS 0.2 μM in a time dependent manner and the level of p21 was remarkably enhanced after 16 hours up to 48 hours incubation. The present study suggested that BCS is a potent inhibitor of the growth of lung cancer cells. Induction of apoptosis related to cell cycle arrest at the G2/M phase with a p53 and p21 dependent pathway (Lee et al 2004).
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