Chen NH, Zhong JJ. Phytomedicine. 2011 Jun 15;18(8-9):719-25. Epub 2011 Feb 25.

The function of p53 induced by ganoderic acids (GAs) in anti-invasion was unknown, although our previous work reported the inhibition of tumor invasion and metastasis by Ganoderic acid T (GA-T). This work indicated that GA-T promoted cell aggregation, inhibited cell adhesion and surpressed cell migration with a dose-dependent manner in human colon tumor cell lines of HCT-116 p53(+/+) and p53(-/-). Furthermore, comparing the ratios of HCT-116 p53(+/+) and p53(-/-) cells, p53 modified GA-T inhibition of migration and adhesion and GA-T promotion of cell aggregation, and p53 also modified GA-T inhibition of NF-κB nuclear translocation, IκBα degradation, and down-regulation of urokinase-type plaminogen activator (uPA), matrix metalloproteinase-2/9 (MMP-2/9), inducible nitric oxide synthase (iNOS/NOS2) protein expression and inducible nitric oxide (NO) production. The results indicated that p53 played an important role in anti-invasion of GA-T in human carcinoma cells. p53 may be an important target for GA-T inhibiting human carcinoma cells anti-invasion.

Ganodermanontriol, a lanostanoid triterpene from Ganoderma lucidum, suppresses growth of colon cancer cells through ß-catenin signaling.

Jedinak A, Thyagarajan-Sahu A, Jiang J, Sliva D. Int J Oncol. 2011 Mar;38(3):761-7. doi: 10.3892/ijo.2011.898. Epub 2011 Jan 11.
Colorectal cancer is one of the most common cancers in men and women in the world. Previous molecular studies have revealed that deregulation of the ß-catenin signaling pathway plays a crucial role in the progression of colorectal cancer. Therefore, modulation of the ß-catenin pathway offers a strategy to control colorectal cancer progression. The medicinal mushroom Ganoderma lucidum (GL) is a rich source of triterpenes with anticancer properties. Here, we show that ganodermanontriol (GNDT), a purified triterpene from GL, inhibited proliferation of HCT-116 and HT-29 colon cancer cells without a significant effect on cell viability. Moreover, GNDT inhibited transcriptional activity of ß-catenin and protein expression of its target gene cyclin D1 in a dose-dependent manner. A marked inhibition effect was also seen on Cdk-4 and PCNA expression, whereas expression of Cdk-2, p21 and cyclin E was not affected by the GNDT treatment. In addition, GNDT caused a dose-dependent increase in protein expression of E-cadherin and ß-catenin in HT-29 cells. Finally, GNDT suppressed tumor growth in a xenograft model of human colon adenocarcinoma cells HT-29 implanted in nude mice without any side-effects and inhibited expression of cyclin D1 in tumors. In conclusion, our data suggest that ganodermanontriol might be a potential chemotherapeutic agent for the treatment of cancer.

Triterpenes from Ganoderma Lucidum induce autophagy in colon cancer through the inhibition of p38 mitogen-activated kinase (p38 MAPK).

Thyagarajan A, Jedinak A, Nguyen H, Terry C, Baldridge LA, Jiang J, Sliva D. Nutr Cancer. 2010;62(5):630-40.
Medicinal mushroom Ganoderma lucidum is one of the most esteemed natural products that have been used in the traditional Chinese medicine. In this article, we demonstrate that G. lucidum triterpene extract (GLT) suppresses proliferation of human colon cancer cells HT-29 and inhibits tumor growth in a xenograft model of colon cancer. These effects of GLT are associated with the cell cycle arrest at G0/G1 and the induction of the programmed cell death Type II-autophagy in colon cancer cells. Here, we show that GLT induces formation of autophagic vacuoles and upregulates expression of Beclin-1 (1.3-fold increase) and LC-3 (7.3-fold increase) proteins in colon cancer cells and in tumors in a xenograft model (Beclin-1, 3.9-fold increase; LC-3, 1.9-fold increase). Autophagy is mediated through the inhibition of p38 mitogen-activated protein kinase (p38 MAPK) because p38 MAPK inhibitor, SB202190, induces autophagy and expression of Beclin-1 (1.2-fold increase) and LC-3 (7.4-fold increase), and GLT suppresses phosphorylation of p38 MAPK ( approximately 60% inhibition) in colon cancer cells. Taken together, our data demonstrate a novel mechanism responsible for the inhibition of colon cancer cells by G. lucidum and suggest GLT as natural product for the treatment of colon cancer.

Ganoderic acid T inhibits tumor invasion in vitro and in vivo through inhibition of MMP expression.

Chen NH, Liu JW, Zhong JJ. Pharmacol Rep. 2010 Jan-Feb;62(1):150-63.
The traditional Chinese medicinal mushroom, Ganoderma lucidum, has been used in Asia for several thousand years for the prevention and treatment of a variety of diseases, including cancer. In previous work, we purified ganoderic acid T (GA-T) from G. lucidum [28]. In the present study, we investigate the functions of GA-T in terms of its effects on invasion in vitro and metastasis in vivo. A trypan blue dye exclusion assay indicates that GA-T inhibits proliferation of HCT-116 cells, a human colon carcinoma cell line. Cell aggregation and adhesion assays show that GA-T promotes homotypic aggregation and simultaneously inhibits the adhesion of HCT-116 cells to the extracellular matrix (ECM) in a dose-dependent manner. Wound healing assays indicate that GA-T also inhibits the migration of HCT-116 cells in a dose-dependent manner, and it suppresses the migration of 95-D cells, a highly metastatic human lung tumor cell line, in a dose- and time-dependent manner. In addition, GA-T inhibits the nuclear translocation of nuclear factor-kappaB (NF-kappaB) and the degradation of inhibitor of kappaB-alpha (IkappaBalpha), which leads to down-regulated expression of matrix metalloproteinase-9 (MMP-9), inducible nitric oxide synthase (iNOS), and urokinase-type plasminogen activator (uPA). Animal and Lewis Lung Carcinoma (LLC) model experiments demonstrate that GA-T suppresses tumor growth and LLC metastasis and down-regulates MMP-2 and MMP-9 mRNA expression in vivo. Taken together, these results demonstrate that GA-T effectively inhibits cancer cell invasion in vitro and metastasis in vivo, and thus it may act as a potential drug for treating cancer.

Effects of Ganoderma lucidum on apoptotic and anti-inflammatory function in HT-29 human colonic carcinoma cells.

Hong KJ, Dunn DM, Shen CL, Pence BC. Phytother Res. 2004 Sep;18(9):768-70.
Ling Zhi extract (LZE) is a herbal mushroom preparation which been used world wide for the prevention and treatment of various cancers. The current study was designed to evaluate these claims in human colon cancer cells in terms of cancer preventive mechanisms. Results have demonstrated induction of apoptosis, anti-inflammatory action and differential cytokine expression during induced inflammation in the human colonic carcinoma cell line, HT-29. LZE caused no cytotoxicity in HT-29 cells at doses less than 10,000 microg/ml. Increasing concentrations of LZE reduced prostaglandin E2 production, but increased nitric oxide production. LZE treatment induced apoptosis by increasing the activity of caspase-3. RT-PCR showed that LZE at a concentration of 5000 microg/ml decreased the expression of cyclooxygenase-2 mRNA. Among 42 cytokines tested by protein array in this study, supplementation of LZE at doses of 500 and 5000 microg/ml to HT-29 cells reduced the expression of interleukin-8, macrophage inflammatory protein 1-delta, vascular epithelial growth factor, and platelet-derived growth factor. These results suggest that LZE has pro-apoptotic and anti-inflammatory functions, as well as inhibitory effects on cytokine expression during early inflammation in colonic carcinoma cells, which may be of significance in the use of Chinese herbal alternative medicines for cancer prevention.

Isoflavone aglycon produced by culture of soybean extracts with basidiomycetes and its anti-angiogenic activity.

Miura T, Yuan L, Sun B, Fujii H, Yoshida M, Wakame K, Kosuna K. Biosci Biotechnol Biochem. 2002 Dec;66(12):2626-31.
Soybean extracts (SBE) containing isoflavone glycosides were cultured with Ganoderma lucidum mycelia producing beta-glucosidase. The anti-angiogenic effects of the cultivated product, containing rich in genistein, named GCP (genistein combined polysaccharide), were assessed with chick chorioallantoic membranes (CAM) and a mouse dorsal air-sac model. Beta-glucosidase produced by the mycelia converted the isoflavone glycosides into aglycons. A test of volunteers showed that serum concentrations of genistein in the subjects treated with GCP (n = 4) at 3 h after administration were significantly higher than those in the subjects treated with SBE (n = 4). GCP inhibited angiogenesis in CAM, and the activity of GCP was greater than that of SBE. GCP inhibited the formation of new vessels induced by colon carcinoma cells in vivo.

A water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia suppresses azoxymethane-induction of colon cancers in male F344 rats.

Lu H, Kyo E, Uesaka T, Katoh O, Watanabe H. Oncol Rep. 2003 Mar-Apr;10(2):375-9.
The present study was designed to investigate the protective effect of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (designated as MAK) on the induction and development of azoxymethane (AOM)-induced colon tumors in male F344/Du Crj rats. A total of 80 animals were divided into five groups at six weeks of age, groups 2, 3 and 4 being given weekly subcutaneous injections of AOM (15 mg/kg body weight) for the initial 3 weeks to induce colon tumors. Rats in group 1 and 5 were injected with the vehicle, 0.9% (w/v) saline, following the same schedule. Rats in groups 1, 2, 3, 4 and 5 were fed MF, MF, 1.25% MAK, 2.5% MAK and 2.5% MAK diets, respectively, starting 1 week before AOM treatment and throughout the six-month experimental period. There were no significant differences in number of ACF, total AC and AC per site among groups 2 to 4, but the tumor incidence was significantly lower, and tumor size was smaller in group 4 (AOM + 2.5% MAK) than in group 2 (AOM + MF). Additionally, beta-catenin positive tumor cell nuclei were significantly decreased in the MAK-fed rats (groups 3 and 4), which also demonstrated lowering of the PCNA labeling index and a shortened germinal region in the colon. The present results thus indicate that dietary MAK could act as a potent chemopreventive agent for colon carcinogenesis.

Prevention of development of N,N’-dimethylhydrazine-induced colon tumors by a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia in male ICR mice.

Lu H, Kyo E, Uesaka T, Katoh O, Watanabe H. Int J Mol Med. 2002 Feb;9(2):113-7.
The protective effects of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (designated as MAK) against development of colon tumors were investigated in male ICR mice. The animals were given weekly injections of N,N’-dimethylhydrazine (DMH, 10 mg/kg body weight) for the initial 10 weeks to induce colon carcinogenesis, and then fed on diet with or without 5% MAK for 10 weeks. There were no significant differences in incidence and the total number of colon tumors between the groups. However, the MAK diet group demonstrated significantly reduced sizes of tumors in comparison with the MF diet group. Moreover, this was linked to a lowered PCNA positive index and shortening of the germinal region in the colon. beta-catenin positive tumor cell nuclei were also significantly decreased in the MAK group. The present results thus indicate that dietary MAK could act as a potent chemopreventive agent for colon carcinogenesis.

Prevention of the development of preneoplastic lesions, aberrant crypt foci, by a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia in male F344 rats.

Lu H, Uesaka T, Katoh O, Kyo E, Watanabe H. Oncol Rep. 2001 Nov-Dec;8(6):1341-5.
The modifying effects of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (MAK) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for three weeks to induce ACF and fed on diets containing 0, 1.25, 2.5 and 5.0% MAK for five weeks, starting one week before the first dose of carcinogen. MAK significantly and dose-dependently prevented the development of ACF, decreasing the total number of AC and inhibiting cyst formation. MAK (2.5 and 5.0%) also significantly reduced the longitudinal-cross section areas of colon epithelium. MAK in all doses significantly reduced the PCNA positive index, area of the germinal region and number of cells per half crypt. In an additional in vitro experiment, MAK inhibited anchorage-independent growth of several colon carcinoma cell lines. The present results thus indicate that dietary MAK could act as a preventive agent for colon carcinogenesis.

Effects of Carvacrol on a Human Non-Small Cell Lung Cancer (NSCLC) Cell Line, A549

Carvacrol, the predominant monoterpene in many essential oils of Labitae including Origanum, Satureja, Thymbra, Thymus, and Corydothymus has substantial antibacterial, antifungal, antihelmintic, insecticial, analgesic and antioxidant activities. Approximately 75% of lung cancer is non-small cell carcinoma (NSCLC) which comprises several histologic types squamous cell, adenocarcinoma and large cell carcinoma. It was reported that the portion of lung tumors diagnosed as denocarcinoma has increased. Thus a human non-small cell lung cancer (NSCLC) cell line, A549 was chosen for this study.
To investigate the effects of carvacrol on cell morphology, apoptosis and total protein amount, the cells incubated with various concentration of carvacrol in DMSO for 24 h. In carvacrol applied A549 cell line, increase in dose of carvacrol caused a decrease in cell number, degeneration of cell morphology and a decrease in total protein amount. To characterize carvacrol induced changes in cell morphology, cells were examined by light microscopy. Cells were treated with carvacrol were seen to have detached from the disk, with cell rounding, cytoplasmic blebbing and irregularity in shape. The data demonstrate that carvacrol is very potent inhibitor of cell growth in A549 cell line.
SOURCE:
Koparal AT, Zeytinoglu M. Cytotechnology. Volume 43, Numbers 1-3 (2003), 149-154, DOI: 10.1023/B:CYTO.0000039917.60348.45

The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland’s morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 μg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and β expression were assessed in paraffin sections. Student’s t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.

We have been studying the gene called RUNX3 and its involvement in human cancer for some time. As will be discussed below, RUNX3 might be one of the genes that may contribute to early diagnosis.
RUNX gene is a mammalian homolog of Drosophila segmentation gene, Runt. The cDNA sequence of Runt was described first by Peter Gergen. Then Misao Ohki identified AML1 gene on chromosome 21 that is involved in chromosomal translocation, t(8;21), which is the most frequently observed chromosome translocation in acute myeloid leukemia. We were studying transcription factors that regulate gene expression of polyomavirus in developmentally regulated fashion. One of them was polyomavirus enhancer binding protein 2 (PEBP2). Nancy Speck identified a transcription factor called Core Binding Factor (CBF) that regulates retrovirus gene expression. PEBP2 and CBF turned out to be identical and they are heterodimeric transcription factors. The cDNA sequences of Runt, AML1 and DNA binding subunit of PEBP2 and CBF are highly related and we now call them as the RUNX family. From the sequence comparison, it was clear that the gene family is a developmental regulator, involved in human cancer and functions as a transcription factor (Figure1). There are three mammalian RUNX genes, called RUNX1, RUNX2 and RUNX3. These genes are evolutionarily very old and regulate cell specification in development. Developmental regulation and cancer are two different sides of the same coin. We are interested in studying these genes, since our primary interest is to uncover the mechanism by which cancer develops.

We began working on RUNX3 because we found its expression in stomach and intestine and there was a hint that this gene may be involved in stomach and colorectal cancer. To study the gene function, we disrupted Runx3 gene in mouse and examined the changes in intestine and stomach. To analyze intestinal phenotype, we study our Runx3+/- mouse side by side with Min mouse. Min mouse is a mouse line in which one of the allele of APC (adenomatous polyposis coli) gene is inactivated. APC gene is a well-known colon cancer gene and most of the colon cancer patients usually have an impaired APC gene. Min mouse is a standard mouse model to study the mechanism by which APC gene induces colon cancer. In human, colon cancer is common but cancer in small intestine is rare, whereas mouse develops intestinal tumors but only rarely in colon. Tumor formation in intestine in mouse is considered to be a model of human colon cancer. Usually, the function of APC gene is studied in one of the inbred mouse strains, C57BL/6. In this strain, intestinal tumors develop in Min mouse in several months. However, in different mouse strains, the same genetic change in APC gene causes tumors to develop very slowly. The reason is that there is a gene that inhibits the tumor growth when APC gene is inactivated. C57BL/6 strain does not have the function of this gene. We studied Min mutation and Runx3 heterozygous mutation in parallel in BALB/c strain. The reason why we used BALB/c strain was because Runx3-/- mice in C57BL/6 strain die soon after birth, whereas significant percentage of BALB/c strain with Runx3-/- genotype can survive to adulthood. Ironically, Runx3-/- mice did not spontaneously develop tumors. Other investigators also observed that biallelic inactivation of well-known tumor suppressor genes some times do not induce tumors. However, we found that both Min mice and Runx3+/- mice developed several small adenomas, premalignant tumors, at around 15 months after birth: tumor morphology and incidence are remarkably similar. Genomic studies showed that adenomas that developed in Min mice showed homozygous mutation in APC gene, while Runx3 locus did not show any change. On the other hand, in adenomas developed in Runx3+/- mice, there was no sign of genetic alteration in APC gene. Instead, both alleles of Runx3 were inactivated by promoter hypermethylation. This is reminiscent of the frequent observations of RUNX3 hypermethylation in human colorectal cancer. Interestingly, analysis of very small adenomas formed in the compound mice showed either biallelic mutation of APC gene without affecting Runx3 gene or biallelic silencing of Runx3 gene without affecting APC gene. These results strongly suggest that heterozygous mutation of both APC and Runx3 genes are nearly equivalent in inducing adenomas in mouse intestine. Importantly, analysis of human sporadic adenomas revealed similar results. We found that Runx3 attenuates the oncogenic Wnt signaling pathway and that the human gastric cancer-derived mutation R122C is deficient in attenuation of Wnt signaling. These are strong evidence of tumor suppressor activity in Runx3. The results clearly indicate that APC gene and Runx3 gene are similar in inducing early stage of cancer development. APC is characterized as a “gatekeeper” of colon carcinogenesis. Runx3 also functions in the early stage of colon carcinogenesis.

We also studied the knockout phenotype in stomach. In this case, we treated mice with chemical carcinogen which is known to induce gastric cancer. When we give mice N-methyl-N- nitrosourea (MNU) for 10 weeks from 8 weeks of age in drinking water, wild type mice will develop cancer in 1 year of age. We used a smaller dose which will not cause tumor development in wild type mice. When Runx3-/- mice were treated similarly, 100% of the mice developed adenocarcinoma. Runx3+/- mice showed intermediate value, namely showing adenoma and dysplasia. These results show that absence of Runx3 makes mice prone to induce gastric cancer.

From the study of intestine and stomach, we found that Runx3 functions at the beginning of cancer development. This notion is consistent with the results obtained from different studies by others. It has been shown that all Runx1, Runx2 and Runx3 genes induce cellular senescence, that is, permanent cessation of cell division. When activation of oncogene, such as Ras oncogene occurs in normal cells, the cell growth would be strongly stimulated. When this happens in normal cells, however, cellular fail-safe mechanism is activated to stop abnormal cell growth, since such aberrant growth of cells would be harmful to a healthy body. Therefore, Runx genes are at least a part of the fail-safe machinery in our body. If Runx genes are inactivated, the cell would lose a fail-safe mechanism and cells in which oncogenic Ras is activated would keep growing. This is the basis of the first step of carcinogenesis. When these cells acquire additional genetic changes, the cells would gradually become cancer.

It has been known that DNA damage response is a barrier for cancer development. That is to say that when DNA, which stores hereditary information and gets transmitted from generation to generation, is damaged by radiation or toxic substance, our body is equipped with multiple levels of repair mechanisms to rectify such damages. It is now known that the DNA damage response is also activated as an anti-cancer barrier in response to oncogenes such as Ras. This is because oncogenes can also induce DNA damage by increasing the rate of DNA replication. Thus, if this system becomes defective, then damaged DNA would remain in the body which would gradually cause the genomic instability. When genomic instability persists, activation of oncogene or inactivation of tumor suppressor genes could be generated to eventually induce cancer. Therefore, DNA damage response is considered to be an essential barrier to prevent carcinogenesis from being initiated. We found that Runx3 is also involved in DNA damage response.

All these evidence support the notion that Runx3 functions at the initial stage of cancer development. Obviously, cells in our body are protected by multi-levels of protective mechanisms. RUNX family genes seem to be a part of this protective machinery to play a role as a barrier to protect normal cells from becoming malignant. We believe this is the basis of the tumor suppressor activity of RUNX3.

Our studies revealed that RUNX3 is one of the genes that will be worthwhile to study for the purpose of identifying clues of very early stage of cancer. One of the points to note is that mutations in RUNX3 gene in various cancers are rare. RUNX3 is frequently inactivated by methylation of the promoter region. Promoter methylation is the structural modification of the DNA in the regulatory region of the gene to control the expression of the gene. Many tumor suppressor genes are known to be inactivated by promoter methylation. When this structural modification occurs, gene expression is silenced. This is functionally equivalent to mutation of the gene to inactivate it. Interestingly, there have been reports that RUNX3 methylation is a good diagnostic tool for a certain type of colon cancer. Technology to detect methylation of genes is improving. Since RUNX3 is a transcription factor which activates or represses target genes, there is a possibility to detect one of the target genes as a surrogate marker for inactivation of RUNX3. Some of them could be useful tool to detect RUNX3 inactivation.

TRIPTERYGIUM WILFORDII HOOK ANTI-INFLAMMATORY PROPERTIES
The most commonly and extended use of Tripterygium wilfordii Hook refers to its anti-inflammatory properties. In Chinese traditional herbal remedies Tripterygium wilfordii Hook was commonly used for these well known anti-inflammatory properties to treat a variety of autoimmune and inflammatory diseases, however and nowadays the discovery of certain anti-cancer properties on some active constituents of Tripterygium wilfordii Hook, as the diterpenoid epoxide triptolide and the quinone triterpene celastrol opened the door to new drugs that can help on the fight against cancer. Both compounds are the subject of multiple clinical and scientific studies on the search for new anti-cancer agents that may help developing new effective anti-carcinogenic drugs.

HOW DOES TRIPTERYGIUM WILFORDII HOOK FIGHT CANCER?
Before we enter into details on how Celastrol from Tripterygium Wilfordii works performing its anti-cancer function, we need to explain a little bit more some points. The anti-cancer properties exerted by Tripterygium Wilfordii are directly related with a protein present in the human body, the P53. P53 is a tumor suppressor protein that plays a very important role in the fight against cancer. The protein P53 regulates the cell life cycle, telling the body cell when it has come its time to die (this process is known by scientists as apoptosis or programmed cell death -see video below-), thus preventing the uncontrolled cell growth, a disease everyone knows by its most common and scary name, cancer.

CELASTROL AND THE PROTEIN P53
Why is Celastrol so important and what is the link with P53 protein? To answer that we first have to understand what is a proteasome inhibitor, because that’s what Celastrol is, a proteasome inhibitor. A proteasome inhibitor is a drug that blocks the action of proteasomes [7], something that breaks down proteins, as our P53 cancer inhibitor protein. So without proteasomes the P53 is safe and sound, being able to continue its natural cancer inhibition role, telling our damaged cells when it has come their time to die.

TRIPTERYGIUM WILFORDII HOOK IN TRADITIONAL CHINESE MEDICINE
In spite Tripterygium wilfordii hook was originally used in traditional Chinese medicine as a natural anti-inflammatory herbal remedy, in 2006 this active constituent, Celastrol, was studied for its anticancer properties, as it showed to be able to induce leukemia cell apoptosis (programmed cell death) [1]. In some studies in vivo using animal tumor tissue samples showed inhibition of the proteasomal activity and induction of apoptosis in human prostate cancer 26S cells after using Celastrol, demonstrating its potential use as a new anti-cancer agent[1].

CELASTROL AND COLON CANCER, PANCREATIC CANCER AND SQUAMOUS CELL CARCINOMA
Celastrol showed also certain anti-cancer properties for the treatment of colon cancer, squamous cell carcinoma and pancreatic cancer cells [6], showing its potential in suppressing invasion and metastasis of cancer cells.
Last but not least, in July 2011, the Department of Biology and Molecular Biology from Montclair State University, started to show a certain interest in Triptolide, another extract from the herb Tripterygium wilfordii Hook that apparently is able to activates the p53 pathway to trigger apoptosis in human breast cancer cell lines.

References:
[1] Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine,” is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Yang H, Chen D, Cui QC, Yuan X, Dou QP. The Prevention Program, Barbara Ann Karmanos Cancer Institute, and Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
[2] Celastrol targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent cytotoxicity in tumor cells.
Chen G, Zhang X, Zhao M, Wang Y, Cheng X, Wang D, Xu Y, Du Z, Yu X.
[3] Celastrol induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria- and Fas/FasL-mediated pathways.
Mou H, Zheng Y, Zhao P, Bao H, Fang W, Xu N.
Department of Medical Oncology, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang Province 310003, People’s Republic of China.
[3] Celastrus-derived celastrol suppresses autoimmune arthritis by modulating antigen-induced cellular and humoral effector responses.
Venkatesha SH, Yu H, Rajaiah R, Tong L, Moudgil KD.
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
[4] Inhibitory action of Celastrol on hypoxia-mediated angiogenesis and metastasis via the HIF-1α pathway.
Huang L, Zhang Z, Zhang S, Ren J, Zhang R, Zeng H, Li Q, Wu G.
Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China.
[5] Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.
Dai Y, Desano J, Tang W, Meng X, Meng Y, Burstein E, Lawrence TS, Xu L.
Department of Radiation Oncology, Division of Radiation and Cancer Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
[6] Celastrol suppresses invasion of colon and pancreatic cancer cells through the downregulation of expression of CXCR4 chemokine receptor.
Yadav VR, Sung B, Prasad S, Kannappan R, Cho SG, Liu M, Chaturvedi MM, Aggarwal BB.
Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.
[7] Wikipedia
[8] Does triptolide induce lysosomal-mediated apoptosis in human breast cancer cells?
Messina ME Jr, Halaby R.
Montclair State University, Department of Biology and Molecular Biology, 1 Normal Avenue, Montclair, NJ 07043, United States
[9] Benefit of an extract of Tripterygium Wilfordii Hook F in patients with rheumatoid arthritis: a double-blind, placebo-controlled study.
Tao X, Younger J, Fan FZ, Wang B, Lipsky PE.
Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland 20892, USA.

ScienceDaily (Aug. 30, 2007) — A drug that shuts down a critical cell-signaling pathway in the most common and aggressive type of adult brain cancer successfully kills cancer stem cells thought to fuel tumor growth and help cancers evade drug and radiation therapy, a Johns Hopkins study shows.
In a series of laboratory and animal experiments, Johns Hopkins scientists blocked the signaling system, known as Hedgehog, with an experimental compound called cyclopamine (GANT61) to explore the blockade’s effect on cancer stem cells that populate glioblastoma multiforme. Cyclopamine has long been known to inhibit Hedgehog signaling.
“Our study lends evidence to the idea that the lack of effective therapies for glioblastoma may be due to the survival of a rare population of cancer stem cells that appear immune to conventional radiation and chemotherapy,” says Charles G. Eberhart, M.D., Ph.D., associate professor of pathology, ophthalmology and oncology, who led the work. “Hedgehog inhibition kills these cancer stem cells and prevents cancer from growing and may thus develop into the first stem cell-directed therapy for glioblastoma.”

Eberhart cautioned that while his study appears to prove the principle of Hedgehog blocking, much work remains before cyclopamine or any similar drug can be tested in patients. Scientists must determine whether the drug can be effectively and safely delivered to the whole body or whether it must go into the brain, and what if any adverse impact on normal stem cells the treatment might cause.
“Once you’ve answered those questions in animals, the next step would be starting phase I clinical trials in humans,” Eberhart said.
The new study adds to the growing evidence that only a small percentage of cancer cells – in this case stem cells – are capable of unlimited self-renewal and that these cells alone power a tumor’s growth.
Eberhart focused on two pathways important to the survival of normal brain stem cells-Hedgehog and Notch-suspecting that brain cancer stem cells cannot live without them.

The Hedgehog gene, first studied in fruit flies, got its name because during embryonic development, the mutated version causes flies to resemble a spiky hedgehog. The pathway plays a major role in controlling normal fetal and postnatal development, and, later in life, helping normal adult stem cells function and proliferate.
The Johns Hopkins scientists first tested 19 human glioblastomas removed during surgery and frozen immediately, and found Hedgehog active in five at the time of tumor removal. They also found Hedgehog activity in four of seven glioblastoma cell lines.
Next, the team used cyclopamine, chemically extracted from corn lilies that grow in the Rocky Mountains, to inhibit Hedgehog in cells lines growing on plastic or as neurospheres, round clusters of stems cells that float in liquid nutrients. This reduced tumor growth in the cell-laden plastic by 40 to 60 percent, and caused the neurospheres to fall apart Complementarywithout any new growth of the cell clusters.
The researchers also pretreated mice with cyclopamine before injecting human glioblastoma cells into their brains, resulting in cancer cells that failed to form tumors in the mice.

Other researchers have shown that radiotherapy fails to kill all cancer stem cells in glioblastomas, apparently because many of these cells can repair the DNA damage inflicted by radiation. The Hopkins team suggests that blocking the Hedgehog pathway with cyclopamine kills these radiation-resistant cancer stem cells.
In previous laboratory experiments, Eberhart used cyclopamine to block Hedgehog using medulloblastoma cells, the most common brain cancer occurring in children.
Along with childhood brain cancers, cyclopamine has shown early promise in treating skin cancer; rhabdomyosarcoma, a muscle tumor; and multiple myeloma, a cancer of the white blood cells in bone marrow.
“What excites me is that we have taken things we learned about Hedgehog signaling in these relatively rare childhood brain tumors and translated them into an even more aggressive adult tumor,” Eberhart said.

More than 10,000 Americans die annually from glioblastomas. Radiation is the standard therapy for the disease, and several years ago, the U.S. Food and Drug Administration approved adding the drug temozolomide to radiotherapy because the combination provided a small survival increase.
“This is an incredibly difficult tumor to treat,” says first author Eli E. Bar, Ph.D., a postdoctoral fellow. “Survival for glioblastoma has not changed much in 30 years. With the addition of temozolomide, survival got bumped from 12 months to 14 or 15 months.”

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small-molecule inhibitor GANT61 (cyclopamine) induces extensive cell death in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R) downregulated Gli1 and Gli2 expression and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480, and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

Cyclopamine (GANT61)
Cyclopamine (11-deoxojervine) is a naturally occurring chemical that belongs to the group of steroidal jerveratrum alkaloids. It is a teratogen isolated from the corn lily (Veratrum californicum) that causes usually fatal birth defects. It can prevent the fetal brain from dividing into two lobes (holoprosencephaly) and cause the development of a single eye (cyclopia). It does so by inhibiting the hedgehog signaling pathway (Hh). Cyclopamine is useful in studying the role of Hh in normal development, and as a potential treatment for certain cancers in which Hh is overexpressed.
Cyclopamine was named for one-eyed lambs which were born to sheep which grazed on wild corn lily at a farm in Idaho. In 1957 the US Department of Agriculture started an eleven-year investigation which led to the identification of cyclopamine as the cause of the birth defect (Herper, 2005)
Herper M. (2005-11-28). “The Curious Case of The One-Eyed Sheep”. Forbes.

In this study, we explored the anti-metastatic effect of Korean red ginseng in human hepatoma SK-Hep1 cells. Analytical data received from the Korean Ginseng Corporation showed that there was a great variation in ginsenoside contents between WKRG and EKRG, most likely due to the different extracting processes. Although the difference in the effect of these two extracts on cell viability was not prominent, there was a great variation in their inhibitory effects on MMP-2/-9 activities. WKRG inhibited MMP-2/-9 activities in a concentration-dependent manner, while EKRG had no significant inhibition on MMP-2/-9 activities at all tested concentrations. Based on the above observation, WKRG was selected to do the follow-up experiments in this paper. We found that WKRG also diminished uPA activity of SK-Hep1 cells. Moreover, WKRG significantly inhibited the invasion (assessed using the transwell assay) and migration (examined by both transwell and wound-healing assays) of SK-Hep1 cells. We further demonstrated that WKRG notably inhibited the protein expressions of MMP-2 and MMP-9 and increased the protein levels of TIMP-1, TIMP-2 and PAI-1. These results indicated that antimetastatic effect of WKRG was related to the inhibition of enzymatically degradative processes of tumor metastasis. To our knowledge, this is the first study that attempted to explore biochemical mechanisms underlying WKRG’s inhibitory effect on the metastasis of SK-Hep1 cells.Panax ginseng is an herb frequently used in traditional oriental medicine for its wide spectrum of medicinal effects such as tonic, immunomodulatory, anticancer, adaptogenic, antiaging, antioxidant, and neuroprotective effects [10].

The major active components of ginseng are ginsenosides. More than forty ginsenosides have, so far, been isolated and each ginsenoside possesses different pharmacological effects [11]. Commercially available Korean ginseng products are classified into fresh ginseng, white ginseng, and red ginseng. White ginseng is fresh ginseng which has been air-dried, while red ginseng is obtained by heating. Although processed differently, both red and white ginseng products are manufactured from 6-year-old fresh ginseng roots; nevertheless Korean red ginseng has higher contents of ginsenosides such as Rh2, Rg3, and Rg5 in comparison to white ginseng [12]. Rh2 is produced from ginsenoside Rg3 through bacterial transformation and belongs to the protopanaxadiol family. Rh2 has attracted considerable attention owing to its potential tumor-inhibitory activity. It constrains cell growth in MCF-7 human breast cancer and SK-Hep1 hepatoma and can induce apoptosis in various cell lines [13]. Ginsenoside Rg3 has been reported to reduce the gelatinolytic activities of MMP-2 and MMP-9 [14]. Other important ginseng saponins with unique chemopreventive actions include Rb2 which may partly contribute to the inhibition of lung tumor metastasis by arresting tumor-associated angiogenesis [15], and Rp1 (a semisynthesized derivative of ginsenoside Rg5) whose anticancer effect is believed to be achieved by strongly inhibiting tumor cell metastasis and viability, presumably through impeding adhesion and vessel formation [16].Cell migration is a complex process involving many types of intracellular and extracellular components and is associated with signaling pathways. Since migration is a critical event in cancer progression and especially in metastasis, the inhibitory effect of WKRG on cell migration was evaluated [4]. We found that WKRG significantly inhibited the migration and invasion of SK-Hep1 human hepatoma cells. Invasion of cancer cells through a coated membrane involves not only ECM degradation, but also the formation of adhesive interactions between cells and the matrix. Therefore, the cell adhesion assay was carried out; however we arrived with the result that WKRG only caused a small reduction in cell adhesion. The slight reduction in adhesion may be associated with the significant decreases in migration and invasion by WKRG treatment.MMPs belong to a family of zinc-dependent endopeptides. They are secreted as inactive proenzymes and are activated by partial proteolytic cleavage. MMP-2, MMP-9 and dominant MMPs are released by most endothelial cells and appear to play important roles in the degradation of type VI collage, a major constituent of basement membrane, in cancer invasion and metastasis [17].

In this study, we observed up to 62.6% and 81.5% downregulations of MMP-2 and MMP-9 activities by WKRG as compared with the control. Although these are not direct inhibitions of enzymatic activity, they seem to be enough to decrease cancer metastasis, based on other studies [18].The expression of uPA has been suggested to play a critical role in local fibrin deposition/dissolution [17]. Conversion of plasminogen into active plasmin by plasminogen activators, such as uPA, is primary for fibrinolysis to occur. Plasmin degrades fibrin and prevents its extracellular deposition. WKRG caused a reduction in cell surface plasmin activity as evidenced by the uPA activity assay. The importance of fibrinolytic system in wound healing has been demonstrated in plasminogen-deficient mouse models, where healing is weakened primarily due to impaired fibrinolysis, a consequence of insufficient plasmin generation [19]. Furthermore, uPA plays prominent roles in cellular migration and is vital during the initial phases of wound healing [20]. We used the wound healing assay in vitro to observe the effect of WKRG on cellular migration. WKRG potently retarded the migration of cells towards the wounded area. The results of this study demonstrated that WKRG inhibited fibrinolysis and cell migration which are vital during the early phase of wound healing.To further explore the mechanisms underlying the anti-metastatic effect of WKRG, we detected the alteration in levels of several proteins by lysing SK-Hep1 cells after they had been incubated in the presence or absence of WKRG, including MMP-2, MMP-9, TIMP-1, TIMP-2, and PAI-1.

There is in vitro evidence that MMPs and TIMPs are critical in determining the invasive potential of proliferating tumor cells. MMP-2 and MMP-9 are necessary for the migration of many normal cell types and tumor cells [21]; on the other hand, TIMPs are believed to play important roles in the inhibition of growth and migration, especially in hepatocellular carcinomas [22]. These documented findings strongly suggest anti-metastatic potential of WKRG, as we have demonstrated that WKRG suppressed MMP-2/-9 and enhanced TIMP-1/-2 expressions in terms of protein levels.PAI-1 is a serine protease inhibitor which inactivates uPA to prevent it from binding to the uPA receptor (uPAR). uPAR is part of the plasminogen activation system which is also involved in the regulation of cell adhesion, migration, and invasion. It can transmit uPA-mediated extracellular signals inside the cell, probably through the association with different types of integrins and ECM components. Thus PAI-1 has the ability to arrest the proteolysis cascade following uPAR activation [23]. Our experiment by Western blot demonstrated that the protein level of PAI-1 was enhanced in a dose-dependent manner by WKRG treatment. In summary of the above results, it is suggested that WKRG’s influences on MMP-2, MMP-9, uPA, PAI-1, TIMP-1, and TIMP-2 expressions may have a synergic suppressive effect on the migration and invasion of tumor cells.In conclusion, we explored the anti-metastatic effects and mechanistic actions of WKRG in human hepatoma SK-Hep1 cells. It was found that WKRG significantly inhibited the invasion and migration of SK-Hep1 cells. Then, we carried forward the study by showing that WKRG notably inhibited the expressions of MMP-2, MMP-9, and uPA and elevated the protein levels of PAI-1, TIMP-1, and TIMP-2. These results not only evidenced anti-metastatic effect of WKRG but also showed that such effect was associated with the inhibition of enzymatically degradative processes of tumor metastasis. The present study suggests that Korean red ginseng may be developed into a promising agent for cancer therapy. As to which ginsenosides are predominantly responsible for the anti-metastatic effect of Korean red ginseng remain primarily unclear and would require further studies.

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We have investigated the effect of phenolic antioxidants on cisplatin-induced cytotoxicity in vero (African Green Monkey Kidney) cells and in rat renal cortical slices in vitro, and on cisplatin-induced nephrotoxicity in rats in vivo. Incubation of cisplatin with vero cells resulted in time- and concentration-dependent cytotoxicity, as characterized by decreased tryphan blue exclusion (TBE) and increased release of lactate dehydrogenase (LDH) into the medium. Cisplatin also caused reduction of glutathione (GSH) in a concentration-dependent manner. In the rat renal cortical slices model, incubation of cisplatin for 120 min caused an increase in malondialdehyde (MDA), a decrease in GSH and inhibited p-aminohippurate (PAH) uptake in a concentration-dependent manner. Among phenolic antioxidants, iso-eugenol (IG) was found to be more active against cisplatin-induced cytotoxicity in vero cells as well as in rat renal cortical slices than eugenol (EG) and dehydrozingerone (DZ). However none of the test compounds were able to arrest the reduction of the GSH content induced by cisplatin in either the vero cells or the renal cortical slice model. Administration of cisplatin (3 mg/kg) i.p. to rats resulted in significant reduction of body weight, and elevation of blood urea nitrogen (BUN) and serum creatinine. Treatment with IG 10 mg/kg i.p. 1 h before cisplatin resulted in partial but significant protection against the cisplatin-induced reduction of body weight, and elevation of BUN and serum creatinine, the protection being 34,46, and 62%, respectively. EG and DZ (10 mg/kg, i.p.) were found to be inactive in vivo. Because IG is a potent free radical scavenger and protects against cisplatin-induced toxicitiy, the present results have many clinical implications in cisplatin chemotherapy and thus warrants further investigation.