Besides that, the relationship between PR and BCRP has also been focus on. Wang et al found there were progesterone response elements on the upstream of BCRP promoter[114] and they note that the identified PRE is exactly the same as the estrogen response element published by Ee et al[117]. They found that PRB is a strong activator of transcription of the BCRP promoter, and PRA represses the PRB activity in the human placental choriocarcinoma BeWo cells. But the real situation in breast cancer may be complex. Because 17β-estradiol can induce PRB expression and down-regulate BCRP expression through posttranscriptional modification[118]; On the other hand, PRA can repress the estrogen receptor activity[113]. So the relationship between progesterone receptor and BCRP needs

Tumor invasiveness, and metastasis, as well as MDR are still great puzzle in the development and treatment of tumors. The interconversion between epithelial and mesenchymal cells (designated as epithelialmesenchymal or mesenchymal-epithelial transition, EMT or MET, respectively) has received special attention and emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) may take part in the above processes. An epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity, invasiveness, elevated

resistance to apoptosis, and greatly increased production of ECM components[119].

MMP-2, MMP-3 and MMP-9, associated with an invasive phenotype[120].

Kalluri R and Weinberg RA divided EMT into three types[119]. Type 1 EMTs can generate mesenchymal cells (primary mesenchyme) that have the potential to subsequently undergo a MET to generate secondary epithelia during implantation, embryogenesis, and organ development. Type 2 EMTs, the program begins as part of a repair-associated event that normally generates fibroblasts and other related cells in order to reconstruct tissues

Type 3 EMTs occur in neoplastic cells that have previously undergone genetic and epigenetic changes, specifically in genes that favor clonal outgrowth and the development of localized tumors. Envidents show that EMT is critically linked with up-regulated invasion, metastasis, and angiogenesis. Figure 1 displays the relationship between EMT and progression of tumors. During the acquisition of EMT characteristics, cells loose epithelial cell–cell junctions, undergo actin cytoskeleton reorganization and decrease in the expression of proteins that promote cell–cell contact such as E-cadherin and β-catenin, and gain in the expression of mesenchymal markers such as vimentin, fibronectin,γ-smooth muscle actin (SMA), N-cadherin as well as increased activity of matrix metalloproteinases (MMPs) like

The modulation of EMT is complicated. Many genes or signal transduction pathways are confirmed to take part in the regulation, such as hepatocyte growth factor (HGF)[121], transforming growth factor beta (TGF-β)[122], epidermal growth factor (EGF)[123], MMP-3[124] and so on. In addition, some transcriptional factors including snail and twist also play

Recent studies have shown an intimate relationship between the EMT phenotype and MDR. Kajiyama et al. found that paclitaxel-resistant ovarian cancer cells showed phenotypic changes consistent with EMT[127]. These results were confirmed in other types of tumors

further data.

**2.6 EMT and MDR**

following trauma and inflammatory injury.

important role in EMT[125,126].

like gemcitabine-resistant pancreatic cancer cells, oxaliplatin-resistant colorectal cancer cells, lapatinib-resistant breast cancer[120].  It has been indicated that mesenchymal-like cancers might be more sensitive to DNA damaging agents such as doxorubicin, whereas epithelial-like cancers are more sensitive to targeted therapies, such as EGFR and HER2 antagonists [129].That may be the reason why mesenchymal-like, basal breast cancers are initially more sensitive to chemotherapy than epithelial-like luminal breast cancers[130]. However, it was discussed that basal, mesenchymal-like breast cancers possibly would be more prone to develop drug resistance. So more works need to do to investigate the links of EMT and MDR.

Fig. 1. The relationship between EMT and progression of tumors. Nomal epithelial cells transform to tumor cells. After EMT,tumor cells invade into surrounding normal tissues and distant organs. Then, MET reverse the cells into epithelial cells in the metastasis.

#### **2.7 Methylation and MDR**

Cancer is known as a genetic disease. Gain, loss, and mutation of genetic information have long been known to contribute to cancer development and progression. It is being increasingly recognized that epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Methylation of CpG dinucleotides is an important pattern of epigenetics.

Methylation can directly interfere with the binding of transcription factors to inhibit replication and/or methyl-CpG binding proteins that can bind to methylated DNA, as well as regulatory proteins to inhibit transcription[131]. The patterns of CpG methylation are specie and tissue specific. The biological machinery of this system comprises a variety of regulatory proteins including DNA methyltransferases, putative demethylases, methyl-CpG binding proteins, histones modifying enzymes and chromatin remodeling complexes. Alterations in DNA methylation participate in the development of some human diseases, including tumor [132].

Then whether DNA methylation takes part in the modulation of MDR? The answer is yes. El-Osta et al. used inhibitors of DNA methyltransferase (5-azacytidine [5aC]) and histone deacetylase (trichostatin A [TSA]) to examine gene transcription, promoter methylation status, and the chromatin determinants associated with the MDR1 promoter and their result displayed that 5aC and TSA induced DNA demethylation, leading to reactivation of methylated MDR1[133] . Nakayama et al. demonstrated the hypomethylation status of the MDR1 promoter region might be a necessary condition for MDR1 gene overexpression and establishment of P-glycoprotein-mediated multidrug resistance in AML patients[134]. Detailed mapping of MDR1 promoter showed that its promoter is always hypermethylated in drug-sensitive cells, while the drugresistant cells have hypomethylated MDR1

Multidrug Resistence and Breast Cancer 145

individual therapeutic activity and at these elevated doses, both compounds exhibited

In order to solve the high toxicity of the first generation modulators, many newer analogs of the first generation are researched which were more potent and considerably less toxic. Analogs of VRP, including dexverapamil (less cardiotoxic R-enantiomer of VRP), emopamil, gallopamil, and Ro11-2933 (a tiapamil analog) which reversed MDR in vitro to a degree equivalent to VRP, but with marginal toxicity in animal models were documented[2]. The non-immunosuppressive analog of CsA, PSC 833, has demonstrated superior MDR reversal efficacy in conjunction with daunorubicin, DOX, vincristine, vinblastine, taxol, or

Although these agents circumvented many of the problems experienced with first generation MDR modulators, when these agents were co-administered with anticancer agents for modulating P-gp-based MDR, they influenced the pharmacokinetics and biodistribution properties of the anticancer drugs, which resulted in increased toxicity to

The third generation modulators of p-gp have recently been developed using structure– activity relationships and combinatorial chemistry approaches. These agents required low doses (in the nanomolar range (20–100 nM)) to achieve effective reversing concentrations in

The cyclopropyldibenzosuberane LY 335979 is the representative and is currently under investigation in phase II clinical trials. This substance is highly effective on P-gp-mediated MDR at the concentration of 0.1–0.2μM and shows a very strong affinity for P-gp[145]. Compared to CsA, LY 335979 is characterized by a 10-fold increased potency, latent modulating activity and a blockade specific for P-gp. Another drug the acridonecarboxamide GF 120918 exihibits similar characteristics to LY 335979, but seems to be more effective than LY 335979[146]. The effective concentration of it is 20–100 nM and is one of the most potent and selective MDR modulators disclosed thus far. Both of them are specific for P-gp-mediated MDR since it does not modulate MRP-mediated resistance. In addition, some bispecific chemosensitizers that block both P-gp and MRP were found, such

In summary, all the modulators of p-gp can be divided into 10 classifications. Table 1

Besides agents targeting P-gp, drugs that inhibit other genes have also been developing. Table 2 displays the selected list of modulators that inhibit other MDR-related genes. Although these agents appear to be well tolerated in combination with anticancer drugs such as DOX, the lack selectivity for the tumor tissue P-GP is still their deficiency which is

Another method of MDR reversal is the use of monoclonal antibodies, several of which can inhibit P-gp-mediated drug efflux in vitro. The monoclonal antibody (mAb) MRK16 is the

selected list the modulators of p-gp of each classification reported.

mitoxantrone in many cell lines in vitro at concentrations of 0.5–2 mM[143].

severe and sometimes life-threatening toxicities[2].

**<IP_ADDRESS> Second generation modulators**

normal organs such as liver and kidney[144].

**<IP_ADDRESS> Third generation modulators**

as VX-710 and VX-853[2].

**3.1.2 Modulators of other genes**

the cause of adversely affect therapy.

**3.2 Immunotherapy of MDR**

vivo.

promoter[135]. Gayatri Sharma et al used methylation-specific PCR to investigate the promoter methylation status of MDR1 in tumor and serum of 100 patients with invasive ductal carcinomas of breast (IDCs) and MDR1 was hypomethylated in 47% tumors and 44% paired serum of IDC patients [136].

The methylation of BCRP has also been focus on. To et al. have shown an active CpG island within the proximal ABCG2 promoter region contributing to inactivation of ABCG2[137]. A follow-up research by Turner et al. demonstrated that ABCG2 expression in multiple myeloma patients and in cell lines is regulated in part by promoter methylation[138].

DNA methylation has been found to anticipate the regulation of other MDR-related genes. Chekhun VF et al. found that the promoter regions of MDR1, GST-pi, genes were highly methylated in MCF-7 cell line but not in its MCF-7/R drug resistant variant. The results suggests that acquirement of doxorubicin resistance of MCF-7 cells is associated with DNA hypomethylation of the promoter regions of the MDR1, GST-pi[139].

#### **3. Strategies to reverse MDR**

Since MDR phenomena have been recognised, the war fighting against it has been continuing. Many strategies have been devised to overcome it and mainly divided into three types: modulators, immunotherapy and genetic therapy.

#### **3.1 Modulators of MDR**

Because P-gp is the best characterized gene conferring MDR and its wide effects, most modulators target for it. So herein, we divide the modulators into two types: modulators targeting P-gp and targeting other genes.

#### **3.1.1 Modulators of P-gp**

Up to now, numerous compounds have been shown to inhibit the drug efflux function of Pgp and therefore, reverse cellular resistance. Since P-gp was first detected in 1976, three generation of modulators are found or synthesis. The process of chemosensitization involves the co-administration of a MDR modulator with an anticancer drug in order to cause enhanced intracellular accumulation via impairing the P-gp function[2].

#### **<IP_ADDRESS> First generation modulators**

The first compounds documented to reverse MDR was verapamil (VRP), one of the calcium channel blocker[140]. Studies displayed that VRP enhanced intracellular accumulation of many anticancer drugs, including DOX in numerous cell lines. Subsequent studies revealed that this MDR reversing character is shared by many other calcium channel blockers, clinically available calcium antagonists, and calmodulin antagonists, such as felodipine and trifluorperazine[141]. Indole alkaloids, the anti-malarial quinine and the anti-arrhythmic quinidine, have also been shown to reverse MDR in vitro in experimental cell lines [142].Cyclosporin A, a commonly used immunosuppressant for organ transplantation, remains one of the most effective first generation of MDR modulators[2].

A number of these first generation MDR modulators, such as VRP and CsA, displayed excellent MDR reversal activities both preclinically and clinically. However, a unique property shared by most first generation modulators is that they are therapeutic agents and typically reverse MDR at concentrations much higher than those required for their

promoter[135]. Gayatri Sharma et al used methylation-specific PCR to investigate the promoter methylation status of MDR1 in tumor and serum of 100 patients with invasive ductal carcinomas of breast (IDCs) and MDR1 was hypomethylated in 47% tumors and 44%

The methylation of BCRP has also been focus on. To et al. have shown an active CpG island within the proximal ABCG2 promoter region contributing to inactivation of ABCG2[137]. A follow-up research by Turner et al. demonstrated that ABCG2 expression in multiple

DNA methylation has been found to anticipate the regulation of other MDR-related genes. Chekhun VF et al. found that the promoter regions of MDR1, GST-pi, genes were highly methylated in MCF-7 cell line but not in its MCF-7/R drug resistant variant. The results suggests that acquirement of doxorubicin resistance of MCF-7 cells is associated with DNA

Since MDR phenomena have been recognised, the war fighting against it has been continuing. Many strategies have been devised to overcome it and mainly divided into three

Because P-gp is the best characterized gene conferring MDR and its wide effects, most modulators target for it. So herein, we divide the modulators into two types: modulators

Up to now, numerous compounds have been shown to inhibit the drug efflux function of Pgp and therefore, reverse cellular resistance. Since P-gp was first detected in 1976, three generation of modulators are found or synthesis. The process of chemosensitization involves the co-administration of a MDR modulator with an anticancer drug in order to cause

The first compounds documented to reverse MDR was verapamil (VRP), one of the calcium channel blocker[140]. Studies displayed that VRP enhanced intracellular accumulation of many anticancer drugs, including DOX in numerous cell lines. Subsequent studies revealed that this MDR reversing character is shared by many other calcium channel blockers, clinically available calcium antagonists, and calmodulin antagonists, such as felodipine and trifluorperazine[141]. Indole alkaloids, the anti-malarial quinine and the anti-arrhythmic quinidine, have also been shown to reverse MDR in vitro in experimental cell lines [142].Cyclosporin A, a commonly used immunosuppressant for organ transplantation,

A number of these first generation MDR modulators, such as VRP and CsA, displayed excellent MDR reversal activities both preclinically and clinically. However, a unique property shared by most first generation modulators is that they are therapeutic agents and typically reverse MDR at concentrations much higher than those required for their

myeloma patients and in cell lines is regulated in part by promoter methylation[138].

hypomethylation of the promoter regions of the MDR1, GST-pi[139].

enhanced intracellular accumulation via impairing the P-gp function[2].

remains one of the most effective first generation of MDR modulators[2].

types: modulators, immunotherapy and genetic therapy.

paired serum of IDC patients [136].

**3. Strategies to reverse MDR**

targeting P-gp and targeting other genes.

**<IP_ADDRESS> First generation modulators**

**3.1 Modulators of MDR**

**3.1.1 Modulators of P-gp**

individual therapeutic activity and at these elevated doses, both compounds exhibited severe and sometimes life-threatening toxicities[2].

#### **<IP_ADDRESS> Second generation modulators**

In order to solve the high toxicity of the first generation modulators, many newer analogs of the first generation are researched which were more potent and considerably less toxic.

Analogs of VRP, including dexverapamil (less cardiotoxic R-enantiomer of VRP), emopamil, gallopamil, and Ro11-2933 (a tiapamil analog) which reversed MDR in vitro to a degree equivalent to VRP, but with marginal toxicity in animal models were documented[2]. The non-immunosuppressive analog of CsA, PSC 833, has demonstrated superior MDR reversal efficacy in conjunction with daunorubicin, DOX, vincristine, vinblastine, taxol, or mitoxantrone in many cell lines in vitro at concentrations of 0.5–2 mM[143].

Although these agents circumvented many of the problems experienced with first generation MDR modulators, when these agents were co-administered with anticancer agents for modulating P-gp-based MDR, they influenced the pharmacokinetics and biodistribution properties of the anticancer drugs, which resulted in increased toxicity to normal organs such as liver and kidney[144].

#### **<IP_ADDRESS> Third generation modulators**

The third generation modulators of p-gp have recently been developed using structure– activity relationships and combinatorial chemistry approaches. These agents required low doses (in the nanomolar range (20–100 nM)) to achieve effective reversing concentrations in vivo.

The cyclopropyldibenzosuberane LY 335979 is the representative and is currently under investigation in phase II clinical trials. This substance is highly effective on P-gp-mediated MDR at the concentration of 0.1–0.2μM and shows a very strong affinity for P-gp[145]. Compared to CsA, LY 335979 is characterized by a 10-fold increased potency, latent modulating activity and a blockade specific for P-gp. Another drug the acridonecarboxamide GF 120918 exihibits similar characteristics to LY 335979, but seems to be more effective than LY 335979[146]. The effective concentration of it is 20–100 nM and is one of the most potent and selective MDR modulators disclosed thus far. Both of them are specific for P-gp-mediated MDR since it does not modulate MRP-mediated resistance. In addition, some bispecific chemosensitizers that block both P-gp and MRP were found, such as VX-710 and VX-853[2].

In summary, all the modulators of p-gp can be divided into 10 classifications. Table 1 selected list the modulators of p-gp of each classification reported.

#### **3.1.2 Modulators of other genes**

Besides agents targeting P-gp, drugs that inhibit other genes have also been developing. Table 2 displays the selected list of modulators that inhibit other MDR-related genes. Although these agents appear to be well tolerated in combination with anticancer drugs such as DOX, the lack selectivity for the tumor tissue P-GP is still their deficiency which is the cause of adversely affect therapy.

#### **3.2 Immunotherapy of MDR**

Another method of MDR reversal is the use of monoclonal antibodies, several of which can inhibit P-gp-mediated drug efflux in vitro. The monoclonal antibody (mAb) MRK16 is the

Multidrug Resistence and Breast Cancer 147

first antibody used for reversing MDR by Hamada and Tsuruo[147]. The results found that MRK16 increased intracellular accumulation and cytotoxicity of vincristine and actinomycin D in some MDR cell lines, but had no effect on doxorubicin cytotoxicity. An increase in the accumulation ofvincristine and actinomycin D was also observed with two other anti-Pgp

The genetic therapy of MDR mainly includes two methods.The first method was established by Gottesman et al.They produced multidrug resistant bone marrow cells by transfecting them with vectors carrying the MDR1 cDNA and this process allowed bone marrow cells to apply a chemotherapeutic regimen at otherwise unacceptable doses, and thus overcoming

The other method is inhibiting MDR proteins including transcriptional/translational inhibition through the introduction of antisense oligonucleotides or ribozymes or RNA interference.Recently, researchers has done many work targeting different genes, such as mdr1, MRP1, BCRP, GCS and so on. These techniques were proved to have considerable effects on overcoming MDR in vitro and in animal models. However, as many of these methods require gene targeting and transfer, they are unlikely to produce any really

In summary, although many approaches have been adopted to battle with MDR, it will be

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MDR[149].

**4. References**

448–450.

Table 1. Selected list of P-gp modulators[149]

Table 2. Selected list of modulator targeting other MDR-related genes

Anti-arrhythmic agent

Acridone carboxamide LY 335979(zosoquidar) GG918 (GF120918) Peptide chemosensitiser

Quinidine Antifungal agent Ketoconazole Sedative Midazolam

Reversin 121 Reversin 205 Anti-oestrogen Tamoxifen

Immunosuppressant Cyclosporin A Valspodar (PSC833) HIV protease inhibitors

Calcium channel blocker

Progesterone antagonist Mifepristone (RU486)

Name inhibitors MRP1[3] MS-209

 tRA 98006 Flavonoids Phytoestrogens Imatinib mesylate Fumitremorgin C

 TAG- 139 GST-pi[150] Clofibrate Ethacrynic acid GSH analogs Gossypol Indomethacin Misonidazole Piriprost Quinones Quercetin Sulfasalazine GCS[91] PDMP PPMP Miglustat

Table 2. Selected list of modulator targeting other MDR-related genes

 XR-9576 (tariquidar) VX-710 (biricodar) Isothiocyanates tRA 98006 Agosterol A Rifampicin NSAIDs

BCRP(ABCG2)[3] GF-120918 (elacridar)

Table 1. Selected list of P-gp modulators[149]

Ritonavir Saquinavir Nelfi navir

Verapamil Bepridil Diltiazem Flunarizine first antibody used for reversing MDR by Hamada and Tsuruo[147]. The results found that MRK16 increased intracellular accumulation and cytotoxicity of vincristine and actinomycin D in some MDR cell lines, but had no effect on doxorubicin cytotoxicity. An increase in the accumulation ofvincristine and actinomycin D was also observed with two other anti-Pgp mAbs, HYB-241 and HYB-612[148].

#### **3.3 Genetic therapy of MDR**

The genetic therapy of MDR mainly includes two methods.The first method was established by Gottesman et al.They produced multidrug resistant bone marrow cells by transfecting them with vectors carrying the MDR1 cDNA and this process allowed bone marrow cells to apply a chemotherapeutic regimen at otherwise unacceptable doses, and thus overcoming MDR[149].

The other method is inhibiting MDR proteins including transcriptional/translational inhibition through the introduction of antisense oligonucleotides or ribozymes or RNA interference.Recently, researchers has done many work targeting different genes, such as mdr1, MRP1, BCRP, GCS and so on. These techniques were proved to have considerable effects on overcoming MDR in vitro and in animal models. However, as many of these methods require gene targeting and transfer, they are unlikely to produce any really significant in vivo applications anytime soon[149].

In summary, although many approaches have been adopted to battle with MDR, it will be for a long time to ovecome it completely.

#### **4. References**

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**10**

*Taiwan*

*1Institute of Nutrition,*

**Multiple Molecular Targets of**

*2Department of Cosmaceutics, College of Pharmacy,*

*China Medical University, Taichung,*

*Antrodia camphorata***: A Suitable Candidate**

Data from cancer registries reported that cancer incidence is increasing every year and cancer claimed the second leading causes of death worldwide, surpassed only by cardiovascular disease. National Cancer Research Institute classified that the breast cancer ranks second as a cause of cancer death in women, followed by lung cancer.  The incidence of breast cancer varies greatly around the world. Lowest breast cancer incidence was observed in less developed countries, whereas highest in the more developed countries.  It is also notable that about 1 in 8 women in the United States (12%) will develop invasive breast cancer of her life time. In 2010, an estimated 207, 090 new cases of invasive breast cancer were expected to be diagnosed in women in the U.S., along with 54, 000 new cases of non-invasive (*in situ*) breast cancer. In addition, approximately 2000 men are expected to be diagnosed with invasive breast cancer in 2010. The survival rate for women diagnosed with localized breast cancer (cancer that has not spread to lymph nodes or other location in outside the breast) is 98%. If the invasive cancer that has spread to nearby or distant lymph nodes or organs, the five years survival is 84% or 23%, respectively. However, the surprising result is the five year relative survival for female

breast cancer patients has improved from 63% in the early 1960's to 90% today.

Women is primary risk factor for developing breast cancer, because, women's naturally have more breast cells than men. The main reason for develop more breast cells in women due to the constant exposure of growth-promoting effects of the female hormones especially, estrogen and progesterone. Aside from being female, age is the most important risk factor for breast cancer. Potentially modifiable risk factors include weight gain after age 18. About 1 out of 8 invasive breast cancer are found in women younger than 45, while 2 out of 3 invasive breast cancer are common in women age 55 or older. Many studies have shown that being over weight adversely affects survival for postmenopausal women with breast

**1. Introduction**

**for Breast Cancer Chemoprevention**

Hsin-Ling Yang1, K.J. Senthil Kumar2 and You-Cheng Hseu2

## **Multiple Molecular Targets of**  *Antrodia camphorata***: A Suitable Candidate for Breast Cancer Chemoprevention**

Hsin-Ling Yang1, K.J. Senthil Kumar2 and You-Cheng Hseu2 *1Institute of Nutrition, 2Department of Cosmaceutics, College of Pharmacy, China Medical University, Taichung, Taiwan*

#### **1. Introduction**

156 Targeting New Pathways and Cell Death in Breast Cancer

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glycoprotein-mediated transport by the acridone carboxamide derivative GG918.

drug-resistant tumor cells as revealed by monoclonal antibodies. Proc Natl Acad

resistance with a monoclonal antibody. Proc Natl Acad Sci U S A. 1992 Jul

Data from cancer registries reported that cancer incidence is increasing every year and cancer claimed the second leading causes of death worldwide, surpassed only by cardiovascular disease. National Cancer Research Institute classified that the breast cancer ranks second as a cause of cancer death in women, followed by lung cancer.  The incidence of breast cancer varies greatly around the world. Lowest breast cancer incidence was observed in less developed countries, whereas highest in the more developed countries.  It is also notable that about 1 in 8 women in the United States (12%) will develop invasive breast cancer of her life time. In 2010, an estimated 207, 090 new cases of invasive breast cancer were expected to be diagnosed in women in the U.S., along with 54, 000 new cases of non-invasive (*in situ*) breast cancer. In addition, approximately 2000 men are expected to be diagnosed with invasive breast cancer in 2010. The survival rate for women diagnosed with localized breast cancer (cancer that has not spread to lymph nodes or other location in outside the breast) is 98%. If the invasive cancer that has spread to nearby or distant lymph nodes or organs, the five years survival is 84% or 23%, respectively. However, the surprising result is the five year relative survival for female breast cancer patients has improved from 63% in the early 1960's to 90% today.

Women is primary risk factor for developing breast cancer, because, women's naturally have more breast cells than men. The main reason for develop more breast cells in women due to the constant exposure of growth-promoting effects of the female hormones especially, estrogen and progesterone. Aside from being female, age is the most important risk factor for breast cancer. Potentially modifiable risk factors include weight gain after age 18. About 1 out of 8 invasive breast cancer are found in women younger than 45, while 2 out of 3 invasive breast cancer are common in women age 55 or older. Many studies have shown that being over weight adversely affects survival for postmenopausal women with breast

Multiple Molecular Targets of *Antrodia camphorata*:

Fig. 1. Molecular targets of *A. camphorata* on various cancers

A Suitable Candidate for Breast Cancer Chemoprevention 159

leukemia HL-60 cells (Hseu et al., 2002; 2004). Thereafter extensive studies have verified the cancer-preventing or anti-cancer properties of *A. camphorata* and their derived pure compounds in various murine models of human cancer cell lines. Both the fruiting bodies and mycelium of *A. camphorata* have potent anti-proliferative activity against various cancers *in vitro* and *in vivo* (Table. 1). Its chemopreventive action against various cancer cells *via* modulating multiple signaling pathways at various cellular levels, the ultimate outcomes of which are apoptosis, call cycle arrest, growth inhibition, anti-angiogenesis and inhibition of metastasis. Figure 1, illustrating the molecular targets modulated by *A. camphorata*.

cancer risk and those women who are more physically active and less to die from the disease than women who are in active. The actual fact that only 20-30% of women are diagnosed with breast cancer has significant family history of breast cancer. However, a women's risk of breast cancer approximately doubles if she has a first-degree relative (mother, sister, and daughter) who has been diagnosed with breast cancer. Apart from the family history of breast cancer, personal breast cancer history also a major risk factor for further onset. For an example, A women with cancer in one breast has a 3 to 4-fold increased risk of developing a new cancer in the other breast or other part of the same breast. This is unlike from first breast cancer recurrence.

Mammography and ultrasonography are still the most effective for women with non-dense and dense breast tissues, respectively. Additionally, MRI, lymphatic mapping, the nipplesparing mastectomy, partial breast irradiation, neoadjuvant systemic therapy, and adjuvant treatment are promising for subgroups of breast-cancer patients. Although, there few drugs are commercially available, the well known tamoxifen can be offered for endocrineresponsive disease, aromatase inhibitors are increasingly used. Assessment of potential molecular targets is now important in primary diagnosis. Tyrosine kinase inhibitors and other drugs with anti-angiogenesis and cancer cell metastasis inhibitors are currently undergoing preclinical investigations. Recent study show the experimental drug iniparib ultimately shrank tumors and increased the time they took to progress, in addition iniparib prolonging survival in women with what's known as triple-negative breast cancers. This type of breast cancer lacks receptors for estrogen and progesterone and doesn't have large quantities of HER-2/neu protein, which the most successful cancer therapies target. This means that may currently available drugs simply won't affect it. Therefore, new class of chemotherapeutic drug that can potentially inhibit the growth of estrogen-nonresponsible breast cancer are highly warranted.

The indigenous Taiwanese medicinal mushroom *A. camphorata (Syn, Antrodia cinnamomea; Taiwanofungus camphoratus)*, locally known as "Niu Chang Chih" is a parasitic fungus grown in the inner cavity on the aromatic tree *Cinnamomum kaneirai* Hay (Lauraceae). This species is endemic to Taiwan and has been widely used as a Chinese folk medicine and functional food. In Taiwanese culture, it is believed that *Niu Chang Chih* is a valuable gift from the haven. Thereby, it claimed "National treasure of Taiwan". This species was first published by Zang & Su (1990). Dr. Su, a residential chemist, knew Niu Cheng Chih" very well from his chemical studies of various medicinal fungus (Wu et al., 1997) *A. camphorata* is starting to attract interest due to their abundant bioactive phytocompounds including, triterpenoids, flavonoids, polysaccharides, maleic/succinic acid, benzenoids and benzoquinone derivatives. The current scientific world's particular interest in *A. camphorata* and its curative properties originated from the realm of traditional practice. Till the dates there are more then three hundred scientific reports were published regarding the therapeutic potential of *A. camphorata* or it's derived pure compounds. Both the fruiting bodies and mycelium of *A. camphorata* has been shown to exhibit a wide range of health promoting benefits for the hepatic, neurological and cardiovascular systems. It also shown to inhibit variety of inflammation, viral infection, oxidative stress, atheroslerosis and the growth of a variety of cancer cells (Ao et al., 2009; Geethangili & Tzeng, 2009).

#### **1.1 Clinical studies on** *A. camphorata* **as an adjuvant therapy for cancer**

The anticancer potential of *A. camphorata* was recognized as early as 2002, when it was shown to inhibit proliferation and enhanced apoptosis in cultured human premyelocytic

cancer risk and those women who are more physically active and less to die from the disease than women who are in active. The actual fact that only 20-30% of women are diagnosed with breast cancer has significant family history of breast cancer. However, a women's risk of breast cancer approximately doubles if she has a first-degree relative (mother, sister, and daughter) who has been diagnosed with breast cancer. Apart from the family history of breast cancer, personal breast cancer history also a major risk factor for further onset. For an example, A women with cancer in one breast has a 3 to 4-fold increased risk of developing a new cancer in the other breast or other part of the same breast. This is

Mammography and ultrasonography are still the most effective for women with non-dense and dense breast tissues, respectively. Additionally, MRI, lymphatic mapping, the nipplesparing mastectomy, partial breast irradiation, neoadjuvant systemic therapy, and adjuvant treatment are promising for subgroups of breast-cancer patients. Although, there few drugs are commercially available, the well known tamoxifen can be offered for endocrineresponsive disease, aromatase inhibitors are increasingly used. Assessment of potential molecular targets is now important in primary diagnosis. Tyrosine kinase inhibitors and other drugs with anti-angiogenesis and cancer cell metastasis inhibitors are currently undergoing preclinical investigations. Recent study show the experimental drug iniparib ultimately shrank tumors and increased the time they took to progress, in addition iniparib prolonging survival in women with what's known as triple-negative breast cancers. This type of breast cancer lacks receptors for estrogen and progesterone and doesn't have large quantities of HER-2/neu protein, which the most successful cancer therapies target. This means that may currently available drugs simply won't affect it. Therefore, new class of chemotherapeutic drug that can potentially inhibit the growth of estrogen-nonresponsible

The indigenous Taiwanese medicinal mushroom *A. camphorata (Syn, Antrodia cinnamomea; Taiwanofungus camphoratus)*, locally known as "Niu Chang Chih" is a parasitic fungus grown in the inner cavity on the aromatic tree *Cinnamomum kaneirai* Hay (Lauraceae). This species is endemic to Taiwan and has been widely used as a Chinese folk medicine and functional food. In Taiwanese culture, it is believed that *Niu Chang Chih* is a valuable gift from the haven. Thereby, it claimed "National treasure of Taiwan". This species was first published by Zang & Su (1990). Dr. Su, a residential chemist, knew Niu Cheng Chih" very well from his chemical studies of various medicinal fungus (Wu et al., 1997) *A. camphorata* is starting to attract interest due to their abundant bioactive phytocompounds including, triterpenoids, flavonoids, polysaccharides, maleic/succinic acid, benzenoids and benzoquinone derivatives. The current scientific world's particular interest in *A. camphorata* and its curative properties originated from the realm of traditional practice. Till the dates there are more then three hundred scientific reports were published regarding the therapeutic potential of *A. camphorata* or it's derived pure compounds. Both the fruiting bodies and mycelium of *A. camphorata* has been shown to exhibit a wide range of health promoting benefits for the hepatic, neurological and cardiovascular systems. It also shown to inhibit variety of inflammation, viral infection, oxidative stress, atheroslerosis and the growth of a variety of

unlike from first breast cancer recurrence.

breast cancer are highly warranted.

cancer cells (Ao et al., 2009; Geethangili & Tzeng, 2009).

**1.1 Clinical studies on** *A. camphorata* **as an adjuvant therapy for cancer**

The anticancer potential of *A. camphorata* was recognized as early as 2002, when it was shown to inhibit proliferation and enhanced apoptosis in cultured human premyelocytic leukemia HL-60 cells (Hseu et al., 2002; 2004). Thereafter extensive studies have verified the cancer-preventing or anti-cancer properties of *A. camphorata* and their derived pure compounds in various murine models of human cancer cell lines. Both the fruiting bodies and mycelium of *A. camphorata* have potent anti-proliferative activity against various cancers *in vitro* and *in vivo* (Table. 1). Its chemopreventive action against various cancer cells *via* modulating multiple signaling pathways at various cellular levels, the ultimate outcomes of which are apoptosis, call cycle arrest, growth inhibition, anti-angiogenesis and inhibition of metastasis. Figure 1, illustrating the molecular targets modulated by *A. camphorata*.

Fig. 1. Molecular targets of *A. camphorata* on various cancers

Table 1. Anti-cancer activity of *A. camphorata* and it's components against various cancer models

Multiple Molecular Targets of *Antrodia camphorata*:

is significant, and therefore

**2.** *A. camphorata* **regulates cell cycle progression**

A Suitable Candidate for Breast Cancer Chemoprevention 161

In mammalian cells, cell cycle progression is tightly coordinated by the cyclin-dependent protein kinases (Cdk1, Cdk2, Cdk4 and Cdk6), regulatory cyclin subunits (cyclin-A, cyclin-B, cyclin-Ds and cyclin-E) and their inhibitors including p21*WAF1* and p27*KIP1* (Athar et al., 2009). Cyclin-E/Cdk1 (Cdc2) complex is the key components of the cell cycle check point pathway that delays mitotic entry in response to stalled replication or DNA damage. In addition to Cdk1, cyclin-Ds/Cdk4/6, and cyclin-A/Cdk2 are also required for G1/S transition and progression through S phase, while cyclin-A,B/Cdk1 complex activates are required for entry into mitosis (Fig. 1). Although, cyclin-B/Cdk1 complex are maintained inactive during interphase through the phosphorylation of Cdk1 at Thr 14 and Tyr 15 residues, which are catalysed by Wee1 and Myt1 (Thomas et al., 2005). Cyclin-D1 is a rate limiting activator for the G1/S transition, another cell cycle check point. The G1/S transition requires the activation of the cyclinD/Cdk4/Cdk6 and cyclin-E/Cdk2 complexs, which in turn phosphorylates the retinoblastoma protein (Rb). The subsequent dissociation of E2Fs from Rb activates a serious of target genes that are required for cell entering S phase (Athar et al., 2009). Rb was the first tumor suppressor gene, which is essentially hypophosphorylated when cells are in G0 and become progressively phosphorylated by G1 phase cyclin/Cdk complexes as cells enter G1, becoming hypophosphorylated on a larger number of serine and thrionine residues as cells advance through the R point., Rb remaines hypophosphorylated through the reminder of the cell cycle. The phosphorylate groups on Rb are removed by the protein phosphatase (PPI) as cells exit mitosis. Therefore, Rb plays a critical role in cell cycle progression as the molecular governor of the R-point transition (Mathhews & Gerritsen, 2010). Besides, Cdk2 activation, entry into mitosis requires nuclear translocation of active Cdc2/cyclin-B1 complexes. Normally, most Cdc2/cyclin-B1 complexes accumulate in the nucleus during prophase while Cdc25C phosphatase activates

them by dephosphorylating Cdc2 on both Thr14 and Tyr15 (Thomas et al., 2005).

Cell cycle kinases are frequently upregulated in human cancer due to the over expression of their cyclin partners or inactivation of the Cdks inhibitors. Indeed, deregulation of cyclin-D1-Rb axis is very common in human cancers as cyclin-D1 accumulation is found in various types of human malignancies including breast, skin, lung, liver etc., and affect cell cycle modulation perhaps its most extensively studied target (Athar et al., 2009). Number of researchers has been reported on the cancer preventive and therapeutic effects of *A. camphorata* in different *in vitro* and *in vivo* test models. As summarized in Table. 1, various parts including mycelium and fruiting body, extracts (ethanol, methanol and ethyl acetate extracts) and chemical ingredients such as polysaccharides, triterpinoids, sesquiterpine, steroids, phenol compounds, adenosine, cordycepin, ergosterol etc., possessed potent anticancer activity. Besides, breast cancer cells are highly resistant to chemotheraphy, and there is still no effective cure for patients with advanced stages of the disease, specifically in cases of hormone-independent cancer (Rao et al., 2011). In addition, the cost of this therapy

The inhibition of breast cancer cell proliferation by *A. camphorata* was strongly associated with cell cycle arrest and/or induction of apoptosis. Exposure of human breast cancer cells (MCF-7) against *A. camphorata* caused significant arrest of cell progression (Fig. 2) in G1 phase (Yang et al., 2006). It was further confirmed by estrogen non-responsive human breast cancer cell line (MDA-MB-231) showed that approximately 70% of *A. camphorata* treated MDA-MB-231 cells were arrested in sub-G1 (Fig. 2) phase (Hseu et al., 2008). It has

Table 1. Anti-cancer activity of *A. camphorata* and it's components against various cancer

models

#### **2.** *A. camphorata* **regulates cell cycle progression**

In mammalian cells, cell cycle progression is tightly coordinated by the cyclin-dependent protein kinases (Cdk1, Cdk2, Cdk4 and Cdk6), regulatory cyclin subunits (cyclin-A, cyclin-B, cyclin-Ds and cyclin-E) and their inhibitors including p21*WAF1* and p27*KIP1* (Athar et al., 2009). Cyclin-E/Cdk1 (Cdc2) complex is the key components of the cell cycle check point pathway that delays mitotic entry in response to stalled replication or DNA damage. In addition to Cdk1, cyclin-Ds/Cdk4/6, and cyclin-A/Cdk2 are also required for G1/S transition and progression through S phase, while cyclin-A,B/Cdk1 complex activates are required for entry into mitosis (Fig. 1). Although, cyclin-B/Cdk1 complex are maintained inactive during interphase through the phosphorylation of Cdk1 at Thr 14 and Tyr 15 residues, which are catalysed by Wee1 and Myt1 (Thomas et al., 2005). Cyclin-D1 is a rate limiting activator for the G1/S transition, another cell cycle check point. The G1/S transition requires the activation of the cyclinD/Cdk4/Cdk6 and cyclin-E/Cdk2 complexs, which in turn phosphorylates the retinoblastoma protein (Rb). The subsequent dissociation of E2Fs from Rb activates a serious of target genes that are required for cell entering S phase (Athar et al., 2009). Rb was the first tumor suppressor gene, which is essentially hypophosphorylated when cells are in G0 and become progressively phosphorylated by G1 phase cyclin/Cdk complexes as cells enter G1, becoming hypophosphorylated on a larger number of serine and thrionine residues as cells advance through the R point., Rb remaines hypophosphorylated through the reminder of the cell cycle. The phosphorylate groups on Rb are removed by the protein phosphatase (PPI) as cells exit mitosis. Therefore, Rb plays a critical role in cell cycle progression as the molecular governor of the R-point transition (Mathhews & Gerritsen, 2010). Besides, Cdk2 activation, entry into mitosis requires nuclear translocation of active Cdc2/cyclin-B1 complexes. Normally, most Cdc2/cyclin-B1 complexes accumulate in the nucleus during prophase while Cdc25C phosphatase activates them by dephosphorylating Cdc2 on both Thr14 and Tyr15 (Thomas et al., 2005).

Cell cycle kinases are frequently upregulated in human cancer due to the over expression of their cyclin partners or inactivation of the Cdks inhibitors. Indeed, deregulation of cyclin-D1-Rb axis is very common in human cancers as cyclin-D1 accumulation is found in various types of human malignancies including breast, skin, lung, liver etc., and affect cell cycle modulation perhaps its most extensively studied target (Athar et al., 2009). Number of researchers has been reported on the cancer preventive and therapeutic effects of *A. camphorata* in different *in vitro* and *in vivo* test models. As summarized in Table. 1, various parts including mycelium and fruiting body, extracts (ethanol, methanol and ethyl acetate extracts) and chemical ingredients such as polysaccharides, triterpinoids, sesquiterpine, steroids, phenol compounds, adenosine, cordycepin, ergosterol etc., possessed potent anticancer activity. Besides, breast cancer cells are highly resistant to chemotheraphy, and there is still no effective cure for patients with advanced stages of the disease, specifically in cases of hormone-independent cancer (Rao et al., 2011). In addition, the cost of this therapy is significant, and therefore

The inhibition of breast cancer cell proliferation by *A. camphorata* was strongly associated with cell cycle arrest and/or induction of apoptosis. Exposure of human breast cancer cells (MCF-7) against *A. camphorata* caused significant arrest of cell progression (Fig. 2) in G1 phase (Yang et al., 2006). It was further confirmed by estrogen non-responsive human breast cancer cell line (MDA-MB-231) showed that approximately 70% of *A. camphorata* treated MDA-MB-231 cells were arrested in sub-G1 (Fig. 2) phase (Hseu et al., 2008). It has

Multiple Molecular Targets of *Antrodia camphorata*:

2006).

(Ghibelli & Diederich, 2010).

**3.** *A. camphorata* **regulates apoptosis and cell survival**

A Suitable Candidate for Breast Cancer Chemoprevention 163

The major strategies of the underlying case of cancer was attributed to accelerated or dysregulated proliferation leading to cellular expansion and accumulation of tissue mass. It is well understood that key regulators of the cell cycle are frequently altered in many tumor types, with a consequent impact on elements of proliferative control such as cell cycle checkpoints and the response to DNA damage. Therefore, modern chemotherapeutic approaches are designed to exploit such aberration to induce cytotoxicity and tumor regression or cytostasis to control tumor progression. Uncontrolled cell proliferation, however, is only part of the picture. Recent cancer research progress has broadened our understanding underlying etiology to encompass aberrant cellular survival, as a consequence of failing to appropriately induce apoptosis or cell death, which are major contributor to the transformed state. Apoptosis, also known as programmed cell death, is a well-regulated and ordered process that occurs both in development and in response to stress to help maintain tissue homeostasis. Environmental and physico-chemicals stimulates accumulation of mutations or carcinogens that critically alter cell proliferation, cell cycle regulation, cell-cell or cell-extra cellular matrix (ECM) interactions, which eventually leads malignancy. However, apoptosis induction helps to prevent malignancies via eliminating damaged cells (Leibowitz & Yu, 2010). Apoptosis occurred *via* multiple pathways, and the extrinsic death receptor-mediated and the intrinsic mitochondrial-mediated cell death pathway (Fig. 3) are the ones better characterized in molecular terms (Fulda & Debatin,

The role of mitochondria as principal crossroad of the apoptotic process and emerged since 1990's, when it was shown that mitochondria of apoptosing cell death (Ghibelli & Diederich, 2010). The intrinsic pathway is characterized by the rapid release of cytochrome *c* from the mitochondrial inner membrane space into the cytosol. This critical event is absolutely required for caspase-dependent cell death (Kasibhatla & Tseng, 2003). The term mitochondrial outer membrane permeabilization (MOMP) was coined (Green & Kroemer, 2004), which indicates release of inter-membrane proteins rather than ion passage. However, the topological features and size concerns questioned about cytochrome *c* release *via* phospholipids transfer protein (PTP). A channel linking the inter-membrane mitochondrial space to the cytosol was sought to explain release of cytochrome *c*. The release of cytochrome c also depended activation of pro-apoptotic proteins such as Bax/Bid, which was stimulated by physico-chemical-induced cell stress. During the stimulation, cytochrome c nucleates the assembly of a multi-protein complex, known as apoptosomes, functionally analog to the DISC, further recruits and activates the other upstream caspases, including caspase-9 and caspase-8. Caspase-8 and caspase-9 converge into proteolytic activation of caspase-3, results cells undergo execution phase of apoptosis and/or cell dismantling

Number of investigations critically characterized the anticancer potential of *A. camphorata*, regarding to the potential cytotoxic effect of various cancer cells derived from different human origins including lung, liver, breast, prostate, colon and oral cells (Table. 1). The viability of these cancer cells was significantly decreased by *A. camphorata* treatment in a dose-dependent manner. However, different cancer cells responded to *A. camphorata* with different sensitivities.  The reasons as to why *A. camphorata* causes G1 arrest in breast cancer cells, but G2/M phase arrest in other cells are still unknown. Therefore, *A. camphorata* has been shown to modulate the major cell cycle mediators at lower microgram concentrations, arresting breast cancer cells at the G1/S phase of the cell cycle. The anti-proliferative activity of *A. camphorata* involves the induction of p21WAFl and p27KIP1 and down-regulation of cyclin-A/D1/E, cdc2, and CDK4 (Hseu et al., 2008).

Fig. 2.  The reasons as to why *A. camphorata* causes G1 arrest in breast cancer cells, but G2/M phase arrest in other cells are still unknown. Therefore, *A. camphorata* has been shown to modulate the major cell cycle mediators at lower microgram concentrations, arresting breast cancer cells at the G1/S phase of the cell cycle. The anti-proliferative activity of *A. camphorata* involves the induction of p21WAFl and p27KIP1 and down-regulation of

Fig. 2. Schematic diagram of *A. camphorata*-induced cell cycle arrest in various cancer cells

cyclin-A/D1/E, cdc2, and CDK4 (Hseu et al., 2008).

#### **3.** *A. camphorata* **regulates apoptosis and cell survival**

The major strategies of the underlying case of cancer was attributed to accelerated or dysregulated proliferation leading to cellular expansion and accumulation of tissue mass. It is well understood that key regulators of the cell cycle are frequently altered in many tumor types, with a consequent impact on elements of proliferative control such as cell cycle checkpoints and the response to DNA damage. Therefore, modern chemotherapeutic approaches are designed to exploit such aberration to induce cytotoxicity and tumor regression or cytostasis to control tumor progression. Uncontrolled cell proliferation, however, is only part of the picture. Recent cancer research progress has broadened our understanding underlying etiology to encompass aberrant cellular survival, as a consequence of failing to appropriately induce apoptosis or cell death, which are major contributor to the transformed state. Apoptosis, also known as programmed cell death, is a well-regulated and ordered process that occurs both in development and in response to stress to help maintain tissue homeostasis. Environmental and physico-chemicals stimulates accumulation of mutations or carcinogens that critically alter cell proliferation, cell cycle regulation, cell-cell or cell-extra cellular matrix (ECM) interactions, which eventually leads malignancy. However, apoptosis induction helps to prevent malignancies via eliminating damaged cells (Leibowitz & Yu, 2010). Apoptosis occurred *via* multiple pathways, and the extrinsic death receptor-mediated and the intrinsic mitochondrial-mediated cell death pathway (Fig. 3) are the ones better characterized in molecular terms (Fulda & Debatin, 2006).

The role of mitochondria as principal crossroad of the apoptotic process and emerged since 1990's, when it was shown that mitochondria of apoptosing cell death (Ghibelli & Diederich, 2010). The intrinsic pathway is characterized by the rapid release of cytochrome *c* from the mitochondrial inner membrane space into the cytosol. This critical event is absolutely required for caspase-dependent cell death (Kasibhatla & Tseng, 2003). The term mitochondrial outer membrane permeabilization (MOMP) was coined (Green & Kroemer, 2004), which indicates release of inter-membrane proteins rather than ion passage. However, the topological features and size concerns questioned about cytochrome *c* release *via* phospholipids transfer protein (PTP). A channel linking the inter-membrane mitochondrial space to the cytosol was sought to explain release of cytochrome *c*. The release of cytochrome c also depended activation of pro-apoptotic proteins such as Bax/Bid, which was stimulated by physico-chemical-induced cell stress. During the stimulation, cytochrome c nucleates the assembly of a multi-protein complex, known as apoptosomes, functionally analog to the DISC, further recruits and activates the other upstream caspases, including caspase-9 and caspase-8. Caspase-8 and caspase-9 converge into proteolytic activation of caspase-3, results cells undergo execution phase of apoptosis and/or cell dismantling (Ghibelli & Diederich, 2010).

Number of investigations critically characterized the anticancer potential of *A. camphorata*, regarding to the potential cytotoxic effect of various cancer cells derived from different human origins including lung, liver, breast, prostate, colon and oral cells (Table. 1). The viability of these cancer cells was significantly decreased by *A. camphorata* treatment in a dose-dependent manner. However, different cancer cells responded to *A. camphorata* with different sensitivities. A crude aqueous extract obtained from the fermented culture broth of *A. camphorata* exerted a potent effect in reducing the viability of different human breast cancer cell lines, including estrogen-responsive MCF-7 and estrogen-nonresponsive MDA-

Multiple Molecular Targets of *Antrodia camphorata*:

epithelial MCF-10A cells were not affected by antrocin treatment.

The morphological changes of human breast cancer (MCF-7) cells after *A. camphorata*  treatment were further investigated to elucidate the underlying process of reduced viability treatment. The typical morphological characteristics for cell apoptosis, such as cell condensation, plasma membrane blabbing and formation of apoptotic bodies, were confirmed under phase contrast microscopy (Yang et al., 2006). The hallmark of cell apoptosis, DNA fragmentation was also demonstrated in *A. camphorata* treated human breast cancer cells including MCF-7 and MDA-MB-231. Caspase-3 activity in *A. camphorata*treated MCF-7 breast cancer cells was shown to be increased, which further confirmed that *A. camphorata* could induce apoptosis in human breast cancer cells (Yang et al., 2006). An addition to *A. camphorata* crude extract, antrocin, a pure compound isolated from the fruiting bodies of *A. camphorata* significantly induced apoptosis in MDA-MB-231 breast cancer cells through the activation of caspase-3 (Rao et al., 2011). *In vivo* study, we showed that the tumor formation in BALB/c-*nu* mice with the implantation of MDA-MB-231 cells could be inhibited by the administration of the aqueous fermented culture broth of *A. camphorata*. The initial tumor development was dose-dependently inhibited by *A. camphorata*. The significant suppression of tumor development was directly demonstrating the *in vivo* anti-tumor effect of *A. camphorata* (Hseu et al., 2008). Comparably there was no

cytotoxic effect was observed in control mice that received *A. camphorata* alone.

The mechanism of apoptosis induced by *A. camphorata* with various cancer cell lines were extensively studied (Table. 1) and demonstrated the relevant pathways of apoptosis (Fig. 3). There is an increase of the Bax/Bcl-2 ratio associated with the apoptosis induced by *A. camphorata* treatment in MCF-7 and MDA-MB-231 cells (Fig. 3). MCF-7 cells exposed to *A.*

A Suitable Candidate for Breast Cancer Chemoprevention 165

In recent years much interest has been focused investigation onto the cancer preventive compositions of *A. camphorata* rather crude extract. There are two review articles available, thus, extensively summarized potent bioactive components, which are present in *A. camphorata* (Ao et al., 2009; Geethangili & Tzeng, 2009). It is believed that the anti-cancer and anti-metastasis properties of this mushroom are derived from its diversified chemical constituents, although it is comprised primarily of two types of compounds: polysaccharides and triterpenes (Shao et al., 2008). Yeh et al. demonstrated that five lanostanes (dehydroeburicoic acid, 15α-acetyl dehydrosulphurenic acid, 24-triene-21-oic acid, dehydrosulphurenic acid and sulphurenic acid) and three ergostane-type triterpenes (zhankuic acid, zhankuic acid-A and zhankuic acid-C) isolated from fruiting bodies of *A. camphorata* exhibits in vitro cytotoxic effect to various cancer cell lines, including MDA-MB-231. Zhankuic acid and suphurenic acid showed significant cytotoxic effect to the human breast cancer cells MDA-MB-231 and MCF-7 with an IC50 value of 25.1 and 89.2; and 57.8 and 357.0 µM, respectively. Indeed, sulphurenic acid is selectively cytotoxic to estrogennonresponsive MDA-MB-231 cells, and the only one compound that does not contain the diene structure with in the rings at position 7 and 9 and instead possesses a single double bond in the rings at the position 8 (Yeh et al., 2009). This result also provides another hallmark that cytotoxic effect of bioactive compounds also depends on structural activity relationship. Antroquinonol, an ubiquinone derivatives, was isolated from the solid state fermented mycelium of *A. camphorata* exhibits cytotoxic effect against MDA-MB-231 and MCF-7 human breast cancer cells with an IC50 value of 2.64 and 2.1 µM, respectively (Lee et al., 2007). A very similar result were obtained from an another pure compound antrocin, isolated from the fruiting bodies of A. camphorata showed the most anti-proliferative effect against MDA-MB-231 and MCF-7 cells (Rao et al., 2011).  Meanwhile, the highest dose of *A. camphorata* (>100 µg/mL) was used to treat normal human endothelial cells for 24 hours, there was no cytotoxic effects were found. Furthermore, the chloroform extracts of fruiting bodies of *A. camphorata* significantly inhibited the growth of human breast cancer (MCF-7) cells with an IC50 value of 65 µM (Rao et al., 2007). This indicated the differential cytotoxic effects of *A. camphorata*  crude extract on different kinds of breast cancer cells, with no harmful effects on normal cells at higher concentrations (Hseu et al., 2002).

Fig. 3. Schematic representation of *A. camphorata*-induced apoptosis in *via* intrinsic and extrinsic pathways

MB-231 cells with an IC50 value of 57 and 136 µg/mL, respectively (Yang et al., 2006; Hseu et al., 2008; Yang et al., 2011). Meanwhile, the highest dose of *A. camphorata* (>100 µg/mL) was used to treat normal human endothelial cells for 24 hours, there was no cytotoxic effects were found. Furthermore, the chloroform extracts of fruiting bodies of *A. camphorata* significantly inhibited the growth of human breast cancer (MCF-7) cells with an IC50 value of 65 µM (Rao et al., 2007). This indicated the differential cytotoxic effects of *A. camphorata*  crude extract on different kinds of breast cancer cells, with no harmful effects on normal

Fig. 3. Schematic representation of *A. camphorata*-induced apoptosis in *via* intrinsic and

extrinsic pathways

cells at higher concentrations (Hseu et al., 2002).

In recent years much interest has been focused investigation onto the cancer preventive compositions of *A. camphorata* rather crude extract. There are two review articles available, thus, extensively summarized potent bioactive components, which are present in *A. camphorata* (Ao et al., 2009; Geethangili & Tzeng, 2009). It is believed that the anti-cancer and anti-metastasis properties of this mushroom are derived from its diversified chemical constituents, although it is comprised primarily of two types of compounds: polysaccharides and triterpenes (Shao et al., 2008). Yeh et al. demonstrated that five lanostanes (dehydroeburicoic acid, 15α-acetyl dehydrosulphurenic acid, 24-triene-21-oic acid, dehydrosulphurenic acid and sulphurenic acid) and three ergostane-type triterpenes (zhankuic acid, zhankuic acid-A and zhankuic acid-C) isolated from fruiting bodies of *A. camphorata* exhibits in vitro cytotoxic effect to various cancer cell lines, including MDA-MB-231. Zhankuic acid and suphurenic acid showed significant cytotoxic effect to the human breast cancer cells MDA-MB-231 and MCF-7 with an IC50 value of 25.1 and 89.2; and 57.8 and 357.0 µM, respectively. Indeed, sulphurenic acid is selectively cytotoxic to estrogennonresponsive MDA-MB-231 cells, and the only one compound that does not contain the diene structure with in the rings at position 7 and 9 and instead possesses a single double bond in the rings at the position 8 (Yeh et al., 2009). This result also provides another hallmark that cytotoxic effect of bioactive compounds also depends on structural activity relationship. Antroquinonol, an ubiquinone derivatives, was isolated from the solid state fermented mycelium of *A. camphorata* exhibits cytotoxic effect against MDA-MB-231 and MCF-7 human breast cancer cells with an IC50 value of 2.64 and 2.1 µM, respectively (Lee et al., 2007). A very similar result were obtained from an another pure compound antrocin, isolated from the fruiting bodies of A. camphorata showed the most anti-proliferative effect against MDA-MB-231 and MCF-7 cells (Rao et al., 2011). Notably, non-tumorigenic breast epithelial MCF-10A cells were not affected by antrocin treatment.

The morphological changes of human breast cancer (MCF-7) cells after *A. camphorata*  treatment were further investigated to elucidate the underlying process of reduced viability treatment. The typical morphological characteristics for cell apoptosis, such as cell condensation, plasma membrane blabbing and formation of apoptotic bodies, were confirmed under phase contrast microscopy (Yang et al., 2006). The hallmark of cell apoptosis, DNA fragmentation was also demonstrated in *A. camphorata* treated human breast cancer cells including MCF-7 and MDA-MB-231. Caspase-3 activity in *A. camphorata*treated MCF-7 breast cancer cells was shown to be increased, which further confirmed that *A. camphorata* could induce apoptosis in human breast cancer cells (Yang et al., 2006). An addition to *A. camphorata* crude extract, antrocin, a pure compound isolated from the fruiting bodies of *A. camphorata* significantly induced apoptosis in MDA-MB-231 breast cancer cells through the activation of caspase-3 (Rao et al., 2011). *In vivo* study, we showed that the tumor formation in BALB/c-*nu* mice with the implantation of MDA-MB-231 cells could be inhibited by the administration of the aqueous fermented culture broth of *A. camphorata*. The initial tumor development was dose-dependently inhibited by *A. camphorata*. The significant suppression of tumor development was directly demonstrating the *in vivo* anti-tumor effect of *A. camphorata* (Hseu et al., 2008). Comparably there was no cytotoxic effect was observed in control mice that received *A. camphorata* alone.

The mechanism of apoptosis induced by *A. camphorata* with various cancer cell lines were extensively studied (Table. 1) and demonstrated the relevant pathways of apoptosis (Fig. 3). There is an increase of the Bax/Bcl-2 ratio associated with the apoptosis induced by *A. camphorata* treatment in MCF-7 and MDA-MB-231 cells (Fig. 3). MCF-7 cells exposed to *A.*

Multiple Molecular Targets of *Antrodia camphorata*:

inhibitors (Talos and Moll, 2010).

suppressor gene.

or compromised.

A Suitable Candidate for Breast Cancer Chemoprevention 167

a possible role of p53 in M phase came from observations that p53 contributes to the control of centrosome duplication and to the prevention of DNA replication is impaired by spindle

In recent reports indicate that both estrogen receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells were exposed to synthetic or natural derived anti-cancer drugs remarkable arrest cell cycle via accumulation in G2/M phase through the inhibition of Akt activity and p53-independent or p53-dependent activation of p53 inducible proteins such as p21/p53R2 /CDKN1A and GADD45A (De Santi et al., 2011; Hahm et al., 2011; Hsieh et al., 2011). It is noteworthy that *A. camphorata* significantly up-regulates p53 tumor suppressor gene in human colorectal carcinoma cells (Lien et al., 2009), and human prostate cancer cell lines (Chen et al., 2007). However, the potential up-regulation of p53 tumor suppressor gene product by *A. camphorata* was yet to be illustrated in human breast cancer cell lines. Our current work is fascinating *A. camphorata* induced up-regulation of p53 in triple negative MDA-MB-231 cells. We believe coming future this result may give vital evidence that anti-tumor activity of *A. camphorata* through the up-regulation of p53 tumor

Metastasis is characterized that the multistep processes by which cancer spreads from the place at which it first arose as a primary tumor to distant location and establish itself in a new site in the body through blood stream or lymphatic system. Metastasis depends on the cancer cells acquiring two separate abilities increased motility and invasiveness cells that metastasize are basically identical to the original tumor. For example if cancer arises in the breast and metastasizes to the lung, the cancer cells in lung are similar breast cancer cells. Normally our body has many safeguards to prevent cells from adverse cancerous effects. However, many cancer cells itself have the ability to overcome these safeguards. In recent years much research has been focused on to understanding how cancer cells are mutated to circumvent the body's defenses and freely travel to another location. In normal tissue, cells adhere both to one another and a mesh of proteins are filled the space between them, this outer membrane proteins are known as extracellular matrix. The connection between cells and extracellular matrix is particularly characterized that from skin, mouth, lung, stomach and other organs. During invasion, cells spreads, it must break away not only from the cells around it, but also from the extracellular matrix. Cells are tightly bonded with cell-to-cell adhesion molecules. These adhesion molecules also allow interactions between numerous proteins on the cells surface. In cancerous cells, the adhesion molecules seem to be missing

Cadherins, a family of Ca2+-dependent intracellular adhesion protein molecules, which playing vital role for connecting two individual cells. Cadherins also regulates cells morphology, motility and hence tumor invasiveness. The cadherin molecules have three major regions. There is an extracellular region that mediates specific adherins, a transmembrane domain that spans the cell membrane, and a cytoplasmic domain that extends into the cell. The normal pattern of E-cadherin, α-, β-, γ- and p20-catenin are strong membraneous staining with localization at the intracellular border of luminal cells. Abnormal (absent, reduced or localized to cell compartments other than cell membrane) expression of E-cadherin and catenin has been reported in various human cancers and has been associated with tumor progression. The degradation of β-catenin involves its

**5.** *A. camphorata* **down-regulates invasion and metastasis**

*camphorata* significantly enhanced Bax protein expression, whereas, Bcl-2 the anti-apototic protein was not affected (Yang et al., 2006). Rao et al. studied that antrocin, a pure compound isolated from the fruting bodies of *A. camphorata* markedly augmented Bax:Bcl-2/Bcl-xL ratio in MDA-MB-231 cells (Rao et al., 2011). In an i*n vivo* model, Bcl-2-positive cells were observed in MDA-MB-231 cells implanted cells mice tumor tissue. Furthermore, TUNEL assay showed that *A. camphorata*-induced Bcl-2 inhibition was directly proportional to apoptotic cells in mice tumor tissue (Hseu et al., 2008). This study also confirmed that *A. camphorata*-induced tumor suppression was mediated by cell-cycle arrest, as evidenced by reduction of cyclin-D and PCNA protein levels in mice tumor tissues. The down-regulation of Bcl-2 expression is known to be involved in the release of cytochrome *c* from mitochondria from the intrinsic pathway (Fig. 2). Further we investigated the *A. camphorata*induced mitochondria membrame permeability and cytochrome *c* release in MCF-7 cells. Cells treated with *A. camphorata* significantly increased cytochrome *c* accumulation in cytoplasm, which supports *A. camphorata*-induced mitochondrial membrane potential. This data also provided another possible mechanism that *A. camphorata*-induced apoptosis was mediated by mitochondrial membrane potential followed by cytochrome *c* release in human breast cancer cells (Yang et al., 2006).

Another important mediator of apoptosis in immune cells is the Fas receptor/ligand signaling system. The critical elements of the Fas pathway that link receptor-ligand interaction and down-stream activation of caspases, including caspase-3, have been identified (Hung et al., 2010). Recent studies also indicate that widely used chemotherapeutic agents induce apoptosis in susceptible cells. Thereby, the chemotherapeutic agents required an alternative mediator. Recent studies revealed that Fas/Fas ligand (FasL) or death ligand/death receptor (DR) can activate the downstream extrinsic apoptotic pathway (Fig. 2). Gene expressions of both Fas/FasL were induced in human hepatoma HepG2 cells by treatment with methanolic extract of mycelium of *A. camphorata*. However, *A. camphorata* treatment dose-dependently inhibits death receptors (DR-4 and DR-5) and TNF-α receptors (TNFR-I and TNFR-II) in HepG2 cells.

Indeed, these results well demonstrate that *A. camphorata* induced apoptosis possibly by involving up-regulation of Fas expression, which promotes the ligation of Fas and FasL and then passes the death message to cytosolic messengers. As a result, procaspase-8 is activated to caspase-8, which triggers the caspase activation cascade. In addition, this study also demonstrates that *A. camphorata*-induced apoptosis was mediated by mitochondrialindependent pathway (Song, 2005).

#### **4.** *A. camphorata* **up-regulates tumour-suppressor genes**

The p53 tumor suppressor gene encodes a multifunctional protein involved in the comphrehensive control of cellular responses to genotoxic stress. p53 mediated tumor suppressor effects are mediated by a variety of mechanisms including cell cycle arrest, apoptosis and cellular senescence that prevent cells with damaged DNA to pass on their genome to progeny. Recently much attention has been focused towards p53, because it is once of the main effectors of cell cycle check point. However, the precise molecular mechanisms of its action are still controversial. Several reports indicate that p53 directly arrest cells in G1 phase in response to DNA damage, thus preventing DNA synthesis from damaged templates (Wahl et al., 1997). Apart from this p53 is involved in regulating the cell cycle at transition of G1/S and G2/M and with in S phase (Talos & Moll, 2010). Evidence for

*camphorata* significantly enhanced Bax protein expression, whereas, Bcl-2 the anti-apototic protein was not affected (Yang et al., 2006). Rao et al. studied that antrocin, a pure compound isolated from the fruting bodies of *A. camphorata* markedly augmented Bax:Bcl-2/Bcl-xL ratio in MDA-MB-231 cells (Rao et al., 2011). In an i*n vivo* model, Bcl-2-positive cells were observed in MDA-MB-231 cells implanted cells mice tumor tissue. Furthermore, TUNEL assay showed that *A. camphorata*-induced Bcl-2 inhibition was directly proportional to apoptotic cells in mice tumor tissue (Hseu et al., 2008). This study also confirmed that *A. camphorata*-induced tumor suppression was mediated by cell-cycle arrest, as evidenced by reduction of cyclin-D and PCNA protein levels in mice tumor tissues. The down-regulation of Bcl-2 expression is known to be involved in the release of cytochrome *c* from mitochondria from the intrinsic pathway (Fig. 2). Further we investigated the *A. camphorata*induced mitochondria membrame permeability and cytochrome *c* release in MCF-7 cells. Cells treated with *A. camphorata* significantly increased cytochrome *c* accumulation in cytoplasm, which supports *A. camphorata*-induced mitochondrial membrane potential. This data also provided another possible mechanism that *A. camphorata*-induced apoptosis was mediated by mitochondrial membrane potential followed by cytochrome *c* release in human

Another important mediator of apoptosis in immune cells is the Fas receptor/ligand signaling system. The critical elements of the Fas pathway that link receptor-ligand interaction and down-stream activation of caspases, including caspase-3, have been identified (Hung et al., 2010). Recent studies also indicate that widely used chemotherapeutic agents induce apoptosis in susceptible cells. Thereby, the chemotherapeutic agents required an alternative mediator. Recent studies revealed that Fas/Fas ligand (FasL) or death ligand/death receptor (DR) can activate the downstream extrinsic apoptotic pathway (Fig. 2). Gene expressions of both Fas/FasL were induced in human hepatoma HepG2 cells by treatment with methanolic extract of mycelium of *A. camphorata*. However, *A. camphorata* treatment dose-dependently inhibits death receptors

Indeed, these results well demonstrate that *A. camphorata* induced apoptosis possibly by involving up-regulation of Fas expression, which promotes the ligation of Fas and FasL and then passes the death message to cytosolic messengers. As a result, procaspase-8 is activated to caspase-8, which triggers the caspase activation cascade. In addition, this study also demonstrates that *A. camphorata*-induced apoptosis was mediated by mitochondrial-

The p53 tumor suppressor gene encodes a multifunctional protein involved in the comphrehensive control of cellular responses to genotoxic stress. p53 mediated tumor suppressor effects are mediated by a variety of mechanisms including cell cycle arrest, apoptosis and cellular senescence that prevent cells with damaged DNA to pass on their genome to progeny. Recently much attention has been focused towards p53, because it is once of the main effectors of cell cycle check point. However, the precise molecular mechanisms of its action are still controversial. Several reports indicate that p53 directly arrest cells in G1 phase in response to DNA damage, thus preventing DNA synthesis from damaged templates (Wahl et al., 1997). Apart from this p53 is involved in regulating the cell cycle at transition of G1/S and G2/M and with in S phase (Talos & Moll, 2010). Evidence for

(DR-4 and DR-5) and TNF-α receptors (TNFR-I and TNFR-II) in HepG2 cells.

**4.** *A. camphorata* **up-regulates tumour-suppressor genes**

breast cancer cells (Yang et al., 2006).

independent pathway (Song, 2005).

a possible role of p53 in M phase came from observations that p53 contributes to the control of centrosome duplication and to the prevention of DNA replication is impaired by spindle inhibitors (Talos and Moll, 2010).

In recent reports indicate that both estrogen receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells were exposed to synthetic or natural derived anti-cancer drugs remarkable arrest cell cycle via accumulation in G2/M phase through the inhibition of Akt activity and p53-independent or p53-dependent activation of p53 inducible proteins such as p21/p53R2 /CDKN1A and GADD45A (De Santi et al., 2011; Hahm et al., 2011; Hsieh et al., 2011). It is noteworthy that *A. camphorata* significantly up-regulates p53 tumor suppressor gene in human colorectal carcinoma cells (Lien et al., 2009), and human prostate cancer cell lines (Chen et al., 2007). However, the potential up-regulation of p53 tumor suppressor gene product by *A. camphorata* was yet to be illustrated in human breast cancer cell lines. Our current work is fascinating *A. camphorata* induced up-regulation of p53 in triple negative MDA-MB-231 cells. We believe coming future this result may give vital evidence that anti-tumor activity of *A. camphorata* through the up-regulation of p53 tumor suppressor gene.

#### **5.** *A. camphorata* **down-regulates invasion and metastasis**

Metastasis is characterized that the multistep processes by which cancer spreads from the place at which it first arose as a primary tumor to distant location and establish itself in a new site in the body through blood stream or lymphatic system. Metastasis depends on the cancer cells acquiring two separate abilities increased motility and invasiveness cells that metastasize are basically identical to the original tumor. For example if cancer arises in the breast and metastasizes to the lung, the cancer cells in lung are similar breast cancer cells. Normally our body has many safeguards to prevent cells from adverse cancerous effects. However, many cancer cells itself have the ability to overcome these safeguards. In recent years much research has been focused on to understanding how cancer cells are mutated to circumvent the body's defenses and freely travel to another location. In normal tissue, cells adhere both to one another and a mesh of proteins are filled the space between them, this outer membrane proteins are known as extracellular matrix. The connection between cells and extracellular matrix is particularly characterized that from skin, mouth, lung, stomach and other organs. During invasion, cells spreads, it must break away not only from the cells around it, but also from the extracellular matrix. Cells are tightly bonded with cell-to-cell adhesion molecules. These adhesion molecules also allow interactions between numerous proteins on the cells surface. In cancerous cells, the adhesion molecules seem to be missing or compromised.

Cadherins, a family of Ca2+-dependent intracellular adhesion protein molecules, which playing vital role for connecting two individual cells. Cadherins also regulates cells morphology, motility and hence tumor invasiveness. The cadherin molecules have three major regions. There is an extracellular region that mediates specific adherins, a transmembrane domain that spans the cell membrane, and a cytoplasmic domain that extends into the cell. The normal pattern of E-cadherin, α-, β-, γ- and p20-catenin are strong membraneous staining with localization at the intracellular border of luminal cells. Abnormal (absent, reduced or localized to cell compartments other than cell membrane) expression of E-cadherin and catenin has been reported in various human cancers and has been associated with tumor progression. The degradation of β-catenin involves its

Multiple Molecular Targets of *Antrodia camphorata*:

al., 2009; Geethangili & Tzeng, 2011).

A Suitable Candidate for Breast Cancer Chemoprevention 169

It was well demonstrated that metastasis is responsible for the majority of failures in cancer treatment, and is the major case of death from cancer. Therefore, chemotherapy is suggested to prevent local recurrence of the primary tumor and the spread of tumor cells (Weng & Yen, 2010). However, commercially available synthetic chemotherapeutic agents have severe side effects. Recent studies demonstrated that phytochemicals derived from plant source potentially prevents cancer metastasis (Sliva, 2008). As I mentioned that *A. camphorata* has been used in traditional Chinese medicine for various ills, including cancer. The mechanisms of action of *A. camphorata* against cancer cells includes inhibition of cell proliferation, induction of apoptosis and suppression of the motility of highly invasive breast cancer cells were intensively studied (Yang et al., 2006; Hseu et al., 2008). Although there are different compounds with various pharmacological activities were extracted from mycelia, fruiting bodies, spores and fermented culture broth of *A. camphorata*. The anticancer and anti-metastatic activities of this medicinal mushroom were primarily relied on its polysaccharide, benzenoids, lignans, diterpenes, triterpinod and steroid components (Ao et

Our recent study was clearly demonstrated that the anti-invasive and anti-metastatic effects of *A. camphorata* against highly metastatic human breast cancer cells (MDA-MB-231) is due to the inhibition of invasion and metastasis regulatory proteins such as MMP-2, MMP-9, uPA, uPA receptor and VEGF through the down-regulation of MAPK/NF-κB signaling pathway (Yang et al., 2011). *A. camphorata* inhibits invasion and metastasis of breast cancer cells not only through the suppression of MMPs/uPA, it also enhance endogenous MMPs/uPA inhibitors, TIMP-1, TIMP-2 and PAI respectively. It is also well understood that *A. camphorata* potentially modulates MAPKs cascades (Geethangili & Tzeng, 2009). Our investigation also revealed that the inhibition of MMPs/uPA due to the down-regulation of MAPK cascades such as ERK1/2, JNK and p38. Further we observed the major MMP's transcription factor NF-κB also significantly inhibited by *A. camphorata* treatment in MDA-

Moreover, there is substantial evidence on the inhibition of MMP-9/MMP-2 and suppression of invasiveness and metastases of cancer cells using various chemopreventive or chemotherapeutic agents (Ho et al., 2002; Abiru et al., 2002). Based on the observation, *A. camphorata* eventually decreased the activity or protein levels of tumor metastasis-related proteins, including MMP-9, MMP-2, uPA, and uPAR, and increase the expression of their endogenous inhibitors, TIMP-1, TIMP-2, and PAI-1, in MDA-MB-231 cells. Therefore, *A.*

Angiogenesis is the development of a new blood supply from an existing vasculature. Normal cells can stimulate new blood vessels to grow. This happens to repair damaged tissue when wounds are healing. Therefore, normal cells have genes that can produce proteins known as angiogenic factors, which switch blood vessels growth on. However, cells also have genes that produce certain molecules called anti-angiogenic factors, which slow

Accumulating evidences indicate that progressive tumor growth is dependent on angiogenesis. It also plays an important role in the growth and spread of cancer. New blood vessels "feed" the cancer cells with oxygen and nutrients, allowing these cells to grow, invade nearby tissue, spread to other parts of the body, and form new colonies of cancer

*camphorata* could be a potential agent for the prevention of breast cancer metastasis.

**6.** *A. camphorata* **down-regulates tumour angiogenesis**

phosphorylation through complex formation with tumor suppressor gene products such as adenomatous polyposis coli (APC), glycogen synthase kinase-3β (GSK3β) and axin (Davidson et al., 2000). In tumor cells, E-caherin is either partly or entirely missing. This allows tumor cells to detach from each other, and from the matrix which holds everything in place. Recent clinical studies reveled that E-cadherin is important to regulate metastasis. Ecadherin-mediated cell-cell adhesion is associated with the progression of many carcinomas, including breast, bladder and squamous head and neck carcinomas (Davidson et al., 2000). Abnormal expression of E-cadherin and catenin is very common in lobular than ductal carcinomas. However, its expression appears not to be involved into the early stage of neoplasia but to correlate with high grade invasive ductal carcinoma (Nakopoulou et al., 2002). Reduced expression of E-cadherin is seen in about 50% of ductal carcinomas of the breast and is associated with high histological grade, nodal metastases and poor prognosis. Addition to cadherins, a number of proteolytic enzymes contribute to the degradation of environmental barriers, such as the extracellular matrix and the basement membrane. Thus, degradation of the extracellular matrix and components of the basement membrane, mediated by the concerted action of proteinases, such as matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), play a critical role in tumor invasion and metastasis (Westermarck & Kahari, 1999).

Increasing expression of MMP-2, MMP-9 and angiogenic cytokine vascular endothelial growth factor (VEGF) in human breast cancer cell lines including estrogen receptor positive (MCF-7), triple negative (MDA-MB-231) and ductal epithelial tumor (T47D) cells has been suggested to be associated with the highly metastatic potential of breast cancer (Shibata et al., 2002; H.S. Lee et al., 2008). The promoter region of MMP-2 contains various cis-acting elements, including potential binding sites for the transcription factors nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1), and stimulatory protein-1 (SP-1) (Lin et al., 2010). NFkB is critically involved in tumor progression through transcriptional regulation of invasion related factors, such as MMP-2/MM-9, uPA and VEGF (Shibata et al., 2002; Yang et al., 2011). In addition, the involvement of mitogen-activated protein kinase (MAPK) pathways in NF-kB activation has been demonstrated to play an important role in tumor metastasis (Yang et al., 2011). Therefore, the inhibition of MMPs- and/or uPA-mediated migration or invasion could be a potential treatment for preventing or inhibiting cancer metastasis.

Once breast cancer has spread beyond the breast and under arm lymph nodes, it is considered a "systemic" disease, meaning that it is necessary to treat the whole body rather than just one particular spot. If breast cancer cells has traveled through the blood stream or lymphatic system, there are likely to be breast cancer cells in many different parts of the body, even scans only shows few spots. Therefore, the treatment that reaches all parts of the body. Chemotheraphy or hormonal therapy, are more suggestible treatment are used to treat metastic breast cancer instead of treatments that just treat one part of the body, just as surgery. In general, a women might be treated with a hormonal theraphy if she has a hormone responsive (estrogen or progesterone receptor positive) tumor, whereas, tumor that are not responsive to hormonal therapy, further choice is chemotherapy. There are many different types of chemotherapy that are used for breast cancer. In recent years, there has been much interest in developing new types of medicines that kill breast cancer cells in new and different way. Some of these drugs are only work against a specific type of breast cancer. To overcome this problem, current drug discovery system more fascinating a target protein or signaling cascades rather than specific type of breast cancer cells.

phosphorylation through complex formation with tumor suppressor gene products such as adenomatous polyposis coli (APC), glycogen synthase kinase-3β (GSK3β) and axin (Davidson et al., 2000). In tumor cells, E-caherin is either partly or entirely missing. This allows tumor cells to detach from each other, and from the matrix which holds everything in place. Recent clinical studies reveled that E-cadherin is important to regulate metastasis. Ecadherin-mediated cell-cell adhesion is associated with the progression of many carcinomas, including breast, bladder and squamous head and neck carcinomas (Davidson et al., 2000). Abnormal expression of E-cadherin and catenin is very common in lobular than ductal carcinomas. However, its expression appears not to be involved into the early stage of neoplasia but to correlate with high grade invasive ductal carcinoma (Nakopoulou et al., 2002). Reduced expression of E-cadherin is seen in about 50% of ductal carcinomas of the breast and is associated with high histological grade, nodal metastases and poor prognosis. Addition to cadherins, a number of proteolytic enzymes contribute to the degradation of environmental barriers, such as the extracellular matrix and the basement membrane. Thus, degradation of the extracellular matrix and components of the basement membrane, mediated by the concerted action of proteinases, such as matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), play a critical role in tumor invasion and

Increasing expression of MMP-2, MMP-9 and angiogenic cytokine vascular endothelial growth factor (VEGF) in human breast cancer cell lines including estrogen receptor positive (MCF-7), triple negative (MDA-MB-231) and ductal epithelial tumor (T47D) cells has been suggested to be associated with the highly metastatic potential of breast cancer (Shibata et al., 2002; H.S. Lee et al., 2008). The promoter region of MMP-2 contains various cis-acting elements, including potential binding sites for the transcription factors nuclear factor-kappa B (NF-κB), activator protein-1 (AP-1), and stimulatory protein-1 (SP-1) (Lin et al., 2010). NFkB is critically involved in tumor progression through transcriptional regulation of invasion related factors, such as MMP-2/MM-9, uPA and VEGF (Shibata et al., 2002; Yang et al., 2011). In addition, the involvement of mitogen-activated protein kinase (MAPK) pathways in NF-kB activation has been demonstrated to play an important role in tumor metastasis (Yang et al., 2011). Therefore, the inhibition of MMPs- and/or uPA-mediated migration or invasion could be a potential treatment for preventing or inhibiting cancer metastasis. Once breast cancer has spread beyond the breast and under arm lymph nodes, it is considered a "systemic" disease, meaning that it is necessary to treat the whole body rather than just one particular spot. If breast cancer cells has traveled through the blood stream or lymphatic system, there are likely to be breast cancer cells in many different parts of the body, even scans only shows few spots. Therefore, the treatment that reaches all parts of the body. Chemotheraphy or hormonal therapy, are more suggestible treatment are used to treat metastic breast cancer instead of treatments that just treat one part of the body, just as surgery. In general, a women might be treated with a hormonal theraphy if she has a hormone responsive (estrogen or progesterone receptor positive) tumor, whereas, tumor that are not responsive to hormonal therapy, further choice is chemotherapy. There are many different types of chemotherapy that are used for breast cancer. In recent years, there has been much interest in developing new types of medicines that kill breast cancer cells in new and different way. Some of these drugs are only work against a specific type of breast cancer. To overcome this problem, current drug discovery system more fascinating a target

protein or signaling cascades rather than specific type of breast cancer cells.

metastasis (Westermarck & Kahari, 1999).

It was well demonstrated that metastasis is responsible for the majority of failures in cancer treatment, and is the major case of death from cancer. Therefore, chemotherapy is suggested to prevent local recurrence of the primary tumor and the spread of tumor cells (Weng & Yen, 2010). However, commercially available synthetic chemotherapeutic agents have severe side effects. Recent studies demonstrated that phytochemicals derived from plant source potentially prevents cancer metastasis (Sliva, 2008). As I mentioned that *A. camphorata* has been used in traditional Chinese medicine for various ills, including cancer. The mechanisms of action of *A. camphorata* against cancer cells includes inhibition of cell proliferation, induction of apoptosis and suppression of the motility of highly invasive breast cancer cells were intensively studied (Yang et al., 2006; Hseu et al., 2008). Although there are different compounds with various pharmacological activities were extracted from mycelia, fruiting bodies, spores and fermented culture broth of *A. camphorata*. The anticancer and anti-metastatic activities of this medicinal mushroom were primarily relied on its polysaccharide, benzenoids, lignans, diterpenes, triterpinod and steroid components (Ao et al., 2009; Geethangili & Tzeng, 2011).

Our recent study was clearly demonstrated that the anti-invasive and anti-metastatic effects of *A. camphorata* against highly metastatic human breast cancer cells (MDA-MB-231) is due to the inhibition of invasion and metastasis regulatory proteins such as MMP-2, MMP-9, uPA, uPA receptor and VEGF through the down-regulation of MAPK/NF-κB signaling pathway (Yang et al., 2011). *A. camphorata* inhibits invasion and metastasis of breast cancer cells not only through the suppression of MMPs/uPA, it also enhance endogenous MMPs/uPA inhibitors, TIMP-1, TIMP-2 and PAI respectively. It is also well understood that *A. camphorata* potentially modulates MAPKs cascades (Geethangili & Tzeng, 2009). Our investigation also revealed that the inhibition of MMPs/uPA due to the down-regulation of MAPK cascades such as ERK1/2, JNK and p38. Further we observed the major MMP's transcription factor NF-κB also significantly inhibited by *A. camphorata* treatment in MDA-MB-231 human breast cancer cell (Yang et al., 2011).

Moreover, there is substantial evidence on the inhibition of MMP-9/MMP-2 and suppression of invasiveness and metastases of cancer cells using various chemopreventive or chemotherapeutic agents (Ho et al., 2002; Abiru et al., 2002). Based on the observation, *A. camphorata* eventually decreased the activity or protein levels of tumor metastasis-related proteins, including MMP-9, MMP-2, uPA, and uPAR, and increase the expression of their endogenous inhibitors, TIMP-1, TIMP-2, and PAI-1, in MDA-MB-231 cells. Therefore, *A. camphorata* could be a potential agent for the prevention of breast cancer metastasis.

#### **6.** *A. camphorata* **down-regulates tumour angiogenesis**

Angiogenesis is the development of a new blood supply from an existing vasculature. Normal cells can stimulate new blood vessels to grow. This happens to repair damaged tissue when wounds are healing. Therefore, normal cells have genes that can produce proteins known as angiogenic factors, which switch blood vessels growth on. However, cells also have genes that produce certain molecules called anti-angiogenic factors, which slow down blood vessel growth (Papetti & Herman, 2002).

Accumulating evidences indicate that progressive tumor growth is dependent on angiogenesis. It also plays an important role in the growth and spread of cancer. New blood vessels "feed" the cancer cells with oxygen and nutrients, allowing these cells to grow, invade nearby tissue, spread to other parts of the body, and form new colonies of cancer