Prostaglandins are formed from arachidonic acid through the activities of cyclooxygenase (COX) and subsequent downstream enzymes. Two closed related forms of COXs were discovered which are identified as COX1 and COX2 and both isoenzymes can transform arachidonic acid into prostaglandins, however, differ in their physiological roles. COX1 is the constitutively expressed enzyme, on the other hand, COX2 is an in- ducible form and is expressed in response to growth factors or physiological stimuli as well as inflammatory resultant in the synthesis of prostaglandins which are mediating the pain and supporting the inflammation process. Furthermore, COX2 also plays a role in angio- genesis among certain cancers and in the developing of Alzheimer’s disease (AD). According to recent studies, COX2 is expressed abundantly in these diseases. Stud- ies also suggest that COX2 inhibitors could effectively reduce the risk of developing Alzheimer’s disease. Re- cent studies indicated that overexpression of COX2 is associated with the development of cancers and a rise of COX2 levels is a common finding of cancer formation. COX2 inhibitors, such as non-steroidal anti-inflamma- tory drugs (NSAID's) play anti-cancer effect and also are able to prevent cancer development according to numerous clinical therapy studies. Therefore, it is a new hope to treat or to prevent cancers through the inhibi- tion of COX2 functions or decreasing COX2 protein ex- pression levels. Nowadays many COX2 inhibitors have been discovered. We ought to search natural COX2 in- hibitors existence in plants and we found that Boehme- ria nivea extract (BNE-101) could effectively decreased COX2 protein expression levels in cancer cell lines. Furthermore, BNE-101 could suppress the production of signal molecule related to tumor cells growth, such asb-catenin, and decrease the activities of growth regu- latory proteins, such as phospho-AKT. These data sug- gested that BNE-101 could play roles to inhibit tumor cells growth. We performed the soft agar assay and proved that BNE-101 could inhibit tumor cells growth in a anchorage-independent growth condition. In this study, we also found that BNE-101 decreased the protein expression levels of P-glycoprotein (P-gp), an efflux

pump, which is located on cell membrane to regulate multidrug resistance (MDR) through recognizing dif- ferent chemotherapeutic agents and transporting them out of membrane. Therefore, BNE-101 has great po- tential on regulating P-gp protein expression levels and reducing drug resistant in cancer cells. Overall, we found that BNE-101 could effectively decrease inflam- mation, inhibit cancer cell growth as well as reduce P-gp proteins mediated MDR. It is therefore BNE-101 is effective in the prevention and treatment of cancer.

Key Words: BNE-101, β-catenin, COX2, P-glycoprotein

Introduction

Multiple lines of studies indicate that inflammation leads to the development of chronic diseases and even the onset of cancer. Animal studies demonstrate that chronic inflammation generates various of cancers and inflammation associated genes accompany with the development of cancer. Moreover, increase expression COX2 is associated with premalignant tissues and malignant cancers and reflects the exis- tence of oncogenes, growth factors, cytokines, and tumor promoters. Therefore, agents inhibit COX2 and inflammation associated genes activities to cure or prevent different types of cancers. Animal studies demonstrate that inhibits COX2 gene transcription or specific inhibitors for COX2 protein activities could effectively prevent cancer formation and growth (4). It is, therefore, COX2 could be a therapeutic target for cancer treatment.

In addition to the COX2, intracellular regulatory protein, AKT/protein kinase B (PKB), is also in- volved in the biological response of cells, including cell growth, division and survival, while phospho- rylated in position 308 and 473 plays a role for the regulation AKT protein activity, and the phospho- rylation in 308 position to play more important role

in the development of cancer. Lots of studies indicate that AKT proteins are overexpressed in a large amount of cancer cells (2), therefore, AKT could be a target for anti-cancer therapy.

Recent studies found that AKT could regulate the expression of glucose regulated protein 78 (GRP78), which is induced under the circumstances of en- doplasmic reticulum (ER) under stress. GRP78 is overexpressed in many cancer cells such as breast cancer, prostate cancer, lung cancer, ovarian cancer and colorectal cancer, where the more GRP78 ex- pressed in cancer cells the worst prognosis and leads to impedance for cancer therapy (6). The result in the blockade the expression of GRP78 indicated that GRP78 can be regulated by AKT. Therefore, AKT is another target in cancer therapy.

Beta-catenin β-catenin protein, a multifunc- tional protein, levels are also overexpressed in many cancer cells and the abnormal expression of such protein in cells caused by the cancer formation, development, survival and recurrence (8). Studies found that β-catenin is also regulated by AKT (5). Therefore, β-catenin is an important target to treat cancer.

It is often encountered resistance (multidrug resistance MDR) problem during chemotherapeutic course and makes cancer cells resistant to different anti-cancer drugs simultaneously. The problem is due to the expression of P-glycoprotein, which recognizes different chemotherapy drugs within the cell and pumps

anti-cancer drugs outside the cell. P-glycoprotein is an adenosine triphosphate (ATP) binding cassette transporter which acts as drug efflux pump and nor- mally expresses in cerebral blood barrier (blood-brain barrier BBB). Recent studies indicated that this protein plays a majority role in drugs resistance in cancer cells (1). Therefore, the decreasing expression levels of P-glycoprotein might diminish drugs resis- tance in cancer cells.

To discover pleiotropic drug is an important goal to treat cancer. Compounds exist in plants might be resource to fit for this goal. We found that BNE-101 inhibits effectiveness on a variety of cancer cell growth, including oral cancer, liver cancer, lung cancer, bone cancer and colorectal cancer. BNE-101 works on cancer cells through pleiotropic mechanisms to regu- lated many cancer related proteins such as, inflam- mation associated protein, COX2, cancer survival protein, AKT, anti-apoptosis protein, GRP78, cancer formation related protein, β-catenin, as well as mul- tidrug resistance protein, P-glycoprotein.

Materials and Methods

Cell Culture

The nasopharyngeal carcinoma cell line, KB, and human osteosarcoma cell line, MG-63, were cultured at 37°C in humidified atmosphere of 5% CO2 using

Dulbecco’s modified Eagle’s medium (DMEM) (In- vitrogen Corporation, N.Y., Waltham, MA, U.S.A) supplemented with 10% fetal bovine serum (FBS) and antibiotics (25 U/ml penicillin and 25 U/ml strepto- mycin).

Soft Agar Assay

Cells were suspended in DMEM supplemented with 10% FBS and 0.3% Agar Noble (BD, Difco, Franklin Lakes, NJ, USA) and plated on a layer of 0.5% Agar Noble. Experiments were performed in 6-well plates with approximately 2,500-10,000 cells per well in triplicates. Colonies were stained with Iodonitro- tetrazolium chloride (INT) solution and counted manually after 2-4 weeks of incubation at 37°C and 5% CO2. Colonies were photographed and quantified

manually. The number of colonies was counted under an inverted light microscope at ×40 magnification. The data are shown as mean number of colonies ± standard error (SE) from six fields of three independent wells.

Western Blot Analysis

Cells were harvested in lysis buffer (50 mM Tris-HCl, pH 8.0/250 mM NaCl/1% NP-40, 2 mM EDTA) containing 1 mM PMSF, 10 ng/ml leupeptin, 50 mM NaF, and 1 mM sodium orthovanadate. Immunoblot analysis was performed with the indicated antibodies. Total proteins were then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and specific protein bands visualized with an enhanced chemiluminescence (ECL) detection system (Amer- sham, Waltham, MA, USA).

Colonies

Results

BNE-101 on Cancer Survival Test

We challenge oral cancer, KB, with different con- centrations of BNE-101 and cells were observed at different time points. We found that BNE-101 effec- tively triggered cancer apoptosis, most significant at the concentration of 2 mg/ml (Fig. 1).

We further tested the role of BNE-101 on dif- ferent cancer cell lines. We found that BNE-101 induced apoptosis in colorectal cancer, (HT-29), he- patocellular carcinoma (Hep3B) and osteosarcoma (U2OS) (Fig. 2)

Anchorage-Independent Growth Inhibited by BNE-101

Soft agar culture assay is the most stringency assay to test the growth of cancer cells. We treated oral cancer cells, KB, with different concentrations of BNE-101 for 14 days to test their ability to grow in soft agar. We found that BNE-101 (BNA) at a concentration of 2 mg/ml most effectively inhibited cancer cell growth (Fig. 3) in which the BN was water extraction and BNA was acetonitrile/water (50/50) extracted.

Molecular Mechanisms of BNE-101 on the Growth and Survival of Cancer Cells

COX2 →

AKT →

pAKT Thr308 →

β-Catenin →

P-glycoprotein →

GRP78 →

pp38 →

MMP9 →

Fig. 4. BNE-101 modulated major growth regulatory proteins expression in KB cells.

Above experiments indicated that BNE-101 could effectively inhibit the growth of cancer cells, and it might regulate many key regulatory proteins cell growth and survival. Oral cancer cells were cul- tured with the treatment of different concentration of BNE-101 and Western blot analysis was per- formed to detect the expression of these key regula- tory proteins. A lot of studies suggested that chronic

GRP78

GRP78 →

GRP78 →

β-Catenin

β-Catenin →

β-Catenin →

Hepatoma

→

→

P-glycoprotein

COX2 →

COX2

→

COX2 →

inflammation might increase the chance of cancer, especially the expression inflammation related pro- tein COX2. We found that cancer cells treated with different concentrations of BNE-101 significant inhibited COX2 protein expression (Fig. 4). This experiment indicated that BNE-101 could be effec- tively suppressed inflammatory reactions.

There are numbers of regulatory proteins within the cell and these proteins control cell growth, such as AKT. We found here that the levels of AKT protein were decreased when oral cancer cells treated with different concentrations of BNE-101 as well as the phosphorylation site at 308 in AKT (activated AKT) was also inhibited. The results indicated that cancer cell growth, division and survival messages were in- hibited by BNE-101 (Fig. 4, AKT and pAKT Thr308). There are studies indicate that AKT may also modu- late GRP78 expression and the more GRP78 protein expression in cancer cells results in poor prognosis. Our experiments suggested that GRP78 protein ex- pression was accompanied with AKT activity where both were inhibited by BNE-101 (Fig. 4, GRP78). Therefore, BNE-101 has great beneficial for cancer prognosis. Lots of studies suggest that AKT regulates the expression of β-catenin, which is a key protein to control cancer development. Our result shown here that BNE-101 could inhibitb β-catenin (Fig. 4, β-catenin). The result suggested that BNE-101 could inhibit cancer development, and it had great potential for the purpose of cancer prevention. Moreover, when cells were under oxidative stress, p38 mitogen-activated protein kinase would be activated. We found here that cells treated with BNE-101 didn't result in oxi-

dation stress (Fig. 4, pp38).

At the late stage of cancer development, cancer cells will invade other tissues. It is due to the secretion of metalloproteinases (MMP) in cancer cells, which decomposites extracellular matrix to facilitate ancer cell invasion. We found that BNE-101 could reduce MMP9 protein expression (Fig. 4, MMP9). We in- ferred here that BNE-101 could reduce the invasive- ness in cancer cells.

During cancer treatment, drug resistance is al- ways a big problem and P-glycoprotein is one of the proteins to be blamed. We found here that BNE-101 could effectively inhibit P-glycoprotein expression (Fig. 4, P-glycoprotein). Therefore, BNE-101 can reduce drug resistance problem during cancer treatment.

BNE-101 Inhibits Growth Rregulation Proteins Expres- sion in Colorectal Cancer, Osteosarcoma and Hepatoma

We tested the effects of BNE-101 on more cancer cell lines, and we found that BNE-101 (2 mg/ml) in- hibited β-catenin, pAKT Thr308, COX2, GRP78 and P-glycoprotein (except HT-29 cells because P-gly- coprotein cannot be detected) proteins in HT-29 and U2OS cells (Fig. 5, HT-29 and U2OS). These data indicated that the growth of these two tumor cells lines could be inhibited by the treatment of BNE-101. However, we found that hepatoma cells (Hep3B) treated with BNE-101 resulting in COX2 expression but not GRP78 expression. Beta-catenin, pAKT Thr308, COX2 and P-glycoprotein proteins were inhibited by BNE-101 (Fig. 5). These data shown that BNE-101 could not suppress COX2 and GRP78 expression in a

32

Hep3B

Fig. 6. Analysis Hep3B cells grow in soft agar with the treat- ment of BNE-101.

short period of time. However, BNE-101 could actually inhibit growth and survival signaling molecules. Therefore, we suggested that BNE-101 could inhibit the growth of hepatoma. To test this hypothesis, we grew hepatoma cells, Hep3B, in soft agar with BNE-101 for 14 days and we found that BNE-101 at a concen- tration of 2 mg/ml could inhibit the growth of hepa- toma cells (Fig. 6).

Discussion

Plant field is a excellent nature library. Among them we employed BNE-101 to block cancer cells growth, and we found that product can effectively inhibit a variety of cancer cell growth, including oral cancer, liver cancer, lung cancer, osteosarcoma, and colorectal cancer. BNE-101 works on cancer cells through pleiotropic mechanisms, including the inhibition on the inflammatory COX2 proteins, cancer development regulation protein, β-catenin, cancer cell survival protein, AKT, anti-apoptotic protein, GRP78 and invasion associated protein, MMP9. The result demonstrated that BNE-101 exerted pleiotropic functions in inhibiting cancer development, growth and survival. Beside cancer, chronic inflammation results in many disorders such as immune, cardiovas- cular and nerve diseases. Inflammation is the major response of immunological system in the organism to defense pathogens. Since BNE-101 can inhibit the inflammation protein, COX2, therefore, we infer BNE-101 can be applied to prevent these diseases. This study is the first description on the tumor suppression by BNE-101 through the inhibition of COX2. The results presented here suggested that BNE-101 served as a therapeutic drug for the treatment of tumors. Over- expression of COX-2 has been observed in many tu- mors, such as oral, colorectal, liver, breast and lung cancers. COX-2 may also play a role in the devel- opment of tumor progression. Many clinical studies have been shown a great therapeutic advantage of employment COX-2 inhibitors to treat cancers. COX-2 up-regulation appears to be mediated by nuclear factor-kappa B. However, BNE-101 inhibits the ac- tivities of nuclear factor-kappa B. This is worthy to

Yang

address in the future study.

AKT signaling have multiple functions in cells including promotes cell survival, proliferation and invasion. By blocking this pathway could impede the proliferation of tumor cells. Therefore, the AKT signal transduction pathway could be a therapeutic target in the cancer treatment. The result is demon- strated here that BNE-101 not only inhibited the to- tal AKT proteins, but also reduced the pAKT form. The decreasing pAKT correlated with the decreasing AKT proteins. This result suggested that BNE-101 suppressed the expression of AKT. However, it still didn't rule out that BNE-101 could direct inhibit the AKT up-stream kinase. AKT works against apoptotic pathways to promote cell survival. Therefore, it plays a key role in the resistance of tumor cells therapy. The result showed here that BNE-101 targeting AKT had great advantage to lead tumor cells entering apoptic pathways.

GRP78 is a multiple functional protein. It is well-known as a unfolded protein response which is activated after ER stress. GRP78 is over-expressed in tumor cells, and may activate AKT pro-curvival pathways. GRP78 could promote tumor proliferation, survival, metastasis and also the resistance of wide variety of therapies in cancer cells. Therefore, GRP78 can serve as a target for cancer therapy. The result found that inhibiting the expression of GRP78 cor- related with the suppression cancer cell proliferation by the treatment of BNE-101. However, there is still unclear how BNE-101 regulates the expression of GRP78.

P-glycoprotein is a plasma membrane ATP binding cassette (ABC) to expel cytotoxic drug. The overexpression of P-glycoprotein mediates the re- sistant of chemotherapy in cancer cells. To reduce the expression of P-glycoprotein has created a less resistance in chemotherapy. The result showed here that BNE-101 was able to suppress the expression of P-glycoprotein. It suggested that tumor cells treated with BNE-101 combined with chemotherapeutic drugs had less resistant of chemotherapy.

BNE-101 is extracted from a plant Boehmeria nivea, and has multiple functions in cancer cells to regulate the inflammatory COX2 proteins, cancer development regulation protein, β-catenin, cancer cell survival protein, AKT, anti-apoptotic protein, GRP78 and chemotherapeutic drug resistant protein, P-glycoprotein. The result demonstrated here that BNE-101 exerted pleiotropic functions in inhibiting cancer development, growth and survival.

Conflict of Interest

The authors have no conflicts of interest to report.

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