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Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model.

AIM
To investigate the effects of piperine, a major pungent alkaloid present in Piper nigrum and Piper longum, on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo, and elucidate the underlying mechanisms.


METHODS
Growth of 4T1 cells was assessed using MTT assay. Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs). A highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity. Piperine was injected into tumors every 3 d for 3 times.


RESULTS
Piperine (35-280 μmol/L) inhibited the growth of 4T1 cells in time- and dose-dependent manners (the IC(50) values were 105 ± 1.08 and 78.52 ± 1.06 μmol/L, respectively, at 48 and 72 h). Treatment of 4T1 cells with piperine (70-280 μmol/L) dose-dependently induced apoptosis of 4T1 cells, accompanying activation of caspase 3. The cells treated with piperine (140 and 280 μmol/L) significantly increased the percentage of cells in G(2)/M phase with a reduction in the expression of cyclin B1. Piperine (140 and 280 μmol/L) significantly decreased the expression of MMP-9 and MMP-13, and inhibited 4T1 cell migration in vitro. Injection of piperine (2.5 and 5 mg/kg) dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg) significantly inhibited the lung metastasis.


CONCLUSION
These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo, and has the potential to be developed as a new anticancer drug.


Introduction
Piperine ( Figure 1) is a major pungent alkaloid present in black pepper (Piper nigrum) and long pepper (Piper longum). It exhibits a wide variety of biological effects. Piperine could inhibit both the drug transporter P-glycoprotein and the major drug-metabolizing enzyme CYP3A4 [1,2] , so it has been used as a bioavailability enhancer with various structurally and therapeutically diverse drugs [3] . Piperine also had antiinflammatory, anti-nociceptive, and anti-arthritic effects by inhibiting the expression of interleukin 6 (IL-6), matrix metalloproteinases 13 (MMP-13) and prostaglandin E2 (PGE2) in an arthritis animal model [4] or by inhibiting tumor necrosis www.nature.com/aps Lai LH et al Acta Pharmacologica Sinica npg noma cells [10] , and the anti-invasive effects of piperine on fibrosarcoma cells also have been demonstrated [11] . The therapeutic properties of piperine with other antitumor drugs were tested in various cell types [12,13] . But there is little known about the anticancer activities of piperine on mammary cancer cells and the underlying mechanism. The 4T1 tumors closely mimics human breast cancer in its anatomical site, immunogenicity and growth characteristics [14] . After sc inoculation in the abdominal mammary fat pad, the primary tumor grows into a nodule with the histology of a high-grade breast cancer and sheds spontaneous systemic metastases. Metastatic growth in the lungs is usually the main cause of death of mice. The pattern and histological appearance of such metastases are similar to what is seen in humans, therefore, this model is suitable for testing the effects of experimental therapies on metastatic disease. In the present study, we observed the effects of piperine on the tumor growth and metastasis of 4T1 breast cancer in vitro and in vivo. And the possible mechanism for the inhibitory effect of piperine on tumor cell growth and migration were investigated. The antitumor efficacy makes piperine a potential candidate for future cancer therapy.
Cell lines 4T1 mouse mammary carcinoma cells were a kind gift from Prof Yang-xin FU (Chicago University, Chicago, IL, USA). NIH3T3 (murine fibroblast cell line) cells were obtained from American Type Culture Collection (ATCC, Manassas, VT, USA). Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). Cultures were maintained in a humidified atmosphere with 5% CO 2 at 37 °C.

Animals
Female BALB/c mice syngeneic to 4T1 cells, aged 5-6 weeks, were purchased from Joint Ventures Sipper BK Experimental Animal Co (Shanghai, China) and housed in a room maintained at constant temperature and humidity under 12-h light and darkness cycle and fed with regular autoclave chow diet with water. All animal usage was conducted according to protocols approved by the Zhejiang University Institutional Animal Care and Use Committee.

MTT assay
The effect of piperine on the growth of 4T1 and NIH3T3 cells was examined using a MTT assay. Cells were subcultured in 96-well plates at a density of 10 3 cells per well with or without piperine (35, 70, 100, 140, and 280 µmol/L) for 24 h, 48 h, or 72 h in a final volume of 200 µL. Then, the medium was removed and 20 µL of MTT (5 mg/mL in PBS) was added to the fresh medium. After 2 h incubation at 37 °C, 100 µL DMSO was added to each well and plates were agitated for 1 min. Spectrophotometric absorbance at 570 nm was measured. The percentage of viability was calculated as the following formula: (viable cells)%=(OD of drug-treated sample/OD of untreated sample)×100.

Apoptosis detection assay
After the treatment with piperine 0, 70, 140, and 280 µmol/L for 24 h, harvested cells were suspended in 100 µL binding buffer (1×) including 1 µL Annexin V-FITC and 10 µL PI for 15 min in dark at room temperature and then 400 µL binding buffer (1×) was added to each sample. The FITC and PI fluorescence were measured through FL-1 filter (530 nm) and FL-2 filter (585 nm) respectively, and 10 000 events were acquired.

Cell cycle analysis
After the treatment with piperine 0, 70, 140, and 280 µmol/L for 24 h, 4T1 cells were collected, washed with cold PBS, fixed in cold 70% ethanol and stored at -20 °C overnight. Cells were then washed again with PBS before staining (100 µg/mL of RNase A, 25 µg/mL of propidium iodide and 0.1% of Triton X-100 in PBS), and incubated at 37 °C for 30 min. Cell cycle analysis was performed on flow cytometer.
Western blot analysis 4T1 cells were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice after the treatment with piperine 0, 70, 140, and 280 µmol/L for 24 h. Lysates were then centrifuged at 14 000×g for 10 min to remove insoluble material. Then a BCA kit (Pierce Biotechnology, Rockford, IL, USA) was used to determine the concentration of lysates. Cell extracts were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat milk. The blot was probed with each antibody against Bcl-2, Bax, cyclin D1, cyclin B1, caspase 3, and β-actin. Then the blot was washed, exposed to horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Signals were detected as we previously described [15] .

Wound healing assay
To study the effects of piperine on cell migration in vitro, a wound healing assay was performed following standard methods [16] . 4T1 cells were seeded in 6-well plates and grown www.chinaphar.com Lai LH et al Acta Pharmacologica Sinica npg until 90% confluent. Cells were then serum starved for 24 h, and a linear wound was created in the confluent monolayer using a 200 µL pipette tip. Cells were then washed with PBS and diluted in RPMI-1640 containing 1% FBS with piperine at a concentration of 70 or 140 µmol/L. Images of the wound surfaces were recorded after 24 h.
RNA isolation and real-time quantitative PCR analysis Total RNA from cells was extracted using TRIzol reagent in accordance with the manufacturer's instructions. Real-time quantitative PCR, using SYBR Green detection chemistry, was performed on a 7500 Real-Time PCR System (Applied Biosystems). Quantitative measurements were determined using the ΔΔCt method and expression of GAPDH was used as the internal control. The sequences of the primers are as shown in Table 1.
Evaluation of antitumor activity of piperine in 4T1 mammary carcinoma models 4T1 harvested from subconfluent cultures were washed once in serum-free medium and resuspended in PBS. Cells (5×10 5 ) in 0.1 mL PBS were implanted subcutaneously into the female BALB/c mice in the abdominal mammary fat pad. After 3 d of implantation, piperines (0, 2.5, and 5 mg/kg) were dissolved in 0.2% DMSO and injected into tumors every three days for three times. The tumor volume was measured every two or three days using the formula V=0.5236×d 1 2 ×d 2 , where d 1 is the shortest diameter, and d 2 is the longest diameter. Tumor tissues for histology examination were collected on d 20, fixed in 10% formalin, and embedded in paraffin. Sections (5 µm thick) were prepared for hematoxylin-eosin staining.
Examination of lung metastases by colonogenic assay Cells (5×10 5 ) in 0.1 mL PBS were implanted subcutaneously into the female BALB/c mice. After 3 d of implantation, piperine (0 and 5 mg/kg) were dissolved in 0.2% DMSO and injected into tumors every three days for three times. Five days after the last therapy, primary 4T1 tumors were eliminated from the experiments. For surgical excision of primary 4T1 tumors, mice were anesthetized, and tumors were resected with sterilized instruments. Wounds were closed with metallic clips. Mice in which primary tumors recurred at the site of the surgical excision were eliminated from the experiments. A colonogenic assay was used to evaluate metastases by 4T1 tumors as we previously described [15] . Briefly, lungs were collected and chopped before being dissociated in RPMI-1640 supplemented with 10% FBS containing 1.5 mg/mL collagenase type D (Sigma, St Louis, MO, USA) for 30 min in 37 °C shaking incubator at 178 r/min speed. Lungs were then plated at various dilutions in the RPMI-1640 supplemented with 10% FBS and 60 µmol/L 6-thioguanine. Individual colonies representing micrometastases were counted after 5-10 d.

Statistical analysis
All experiments were repeated two or three times. Data was described as the mean±SD, and statistical analysis was carried out using Student's t-test or the log-rank test (for survival analysis). The difference was considered statistically significant when the P-value was less than 0.05.

Results
Piperine inhibited the growth of 4T1 cells in vitro Inhibitory effects of piperine on the growth of 4T1 cells were examined using the MTT assay. We also tested the effects of piperine on murine NIH3T3 fibroblast cell growth. Compared to the untreated cells (taken as 100% viable), different concentrations of piperine showed a time-and dosedependent inhibition on 4T1 cell growth in the concentration range of 35~280 µmol/L ( Figure 2A). Exposure of 4T1 cells to piperine at 140 µmol/L and 280 µmol/L for 24 h caused a marked decrease in the viability from 100% to 78% and 48% of untreated control levels, respectively. And 48 h of piperine treatment at 140 µmol/L and 280 µmol/L caused a decrease in the viability from 100% to 33% and 18%, respectively. The IC 50 values for piperine were 105±1.08 µmol/L for 48 h, and 78.52±1.06 µmol/L for 72 h treatment ( Figure 2C), suggesting a dose-dependent inhibition of growth, while piperine was not so toxic to NIH3T3 cells ( Figure 2B), for the IC 50 values was 232±1.15 µmol/L for 48 h treatment ( Figure 2C).

Piperine induced apoptosis of 4T1 cells and increased the caspase 3 activity
In order to evaluate the mechanism of growth inhibition of 4T1 cells by piperine, apoptosis of 4T1 cells treated with various concentrations of piperine (0, 140, and 280 µmol/L) for 24 h was analyzed by Annexin V-FITC/PI double-labeled flow cytometry. The apoptosis rate was the sum of early apoptosis and late apoptosis. There was little binding of Annexin-V in untreated and 70 µmol/L piperine treated 4T1 cells ( Figure  3A). But after treatment with piperine at 140 and 280 µmol/L for 24 h, the apoptosis rate was about 24.2%, 23.6%, respectively ( Figure 3B).
Apoptosis is characterized by changes in the expression and activity of several apoptotic markers. The control and regula-   Figure  3C). Caspases are responsible for cell apoptosis. Therefore we determined the effect of piperine on the expression of caspases by Western blot analysis. The results indicated that caspase 3 activity was up-regulated after piperine treatment ( Figure 3C).
Piperine caused the accumulation of the cells in G 2 /M phase After incubation with various concentrations of piperine (0, 70, 140, and 280 µmol/L) for 24 h, cell cycle distributions were analyzed by FCM. We found that with the dose of piperine increased, the percentage of cells in G 2 /M phase was increased, and there were slight changes in G 0 /G 1 phase cell population ( Figure 4A). After incubation with 140 and 280 µmol/L piperine for 24 h, percentage of cells in G 2 /M phase was increased to 7.3%, 15.5%, respectively ( Figure 4B).

Piperine down-regulated the expression of cyclin B1
Given that cyclins are required for the cell cycle progression, the protein levels of cyclin D1 and cyclin B1 in 4T1 cells were detected by Western blot. The result showed that piperine treatment down-regulated the expression of cyclin B1 in a dose-dependent manner, with maximum suppression observed at 280 µmol/L ( Figure 4C), while there was no change of cyclin D1 expression after piperine treatment with various concentrations.

Piperine inhibited the migration ability of 4T1 cells in vitro
We investigated the effects of piperine on the migration properties of 4T1 cells in vitro by wound healing assay. Cells were treated by piperine at a concentration of 0, 70, and 140 µmol/L, and the results showed that piperine inhibited the migration of 4T1 cells in a dose-dependent manner ( Figure 5A).
Piperine decreased the mRNA expression of MMP-9 and 13 Matrix metalloproteinases (MMPs), comprise a family of zinc-containing endopeptidases that share common structural domains. These proteins have the capacity to degrade extracellular matrix (ECM) components, as well as alter their biological functions. Therefore, we examined the mRNA expression of MMP-9, 13, and 14 in 4T1 cells by real-time quantitative PCR after piperine treatment. The mRNA expression of MMP-9 and 13 in 4T1 cells, but not MMP-14, were decreased in a dose-dependent manner ( Figure 5B).

Piperine suppressed 4T1 tumor growth in vivo
To test the antitumor effect of piperine, female BALB/c mice were inoculated sc with 5×10 5 4T1 cells. Piperines (0, 2.5, and 5 mg/kg) were injected intratumorally every three days for three times. The result showed that piperine exhibited a significant inhibition of tumor growth especially at a high dose (5 mg/kg) ( Figure 6A). To determine whether the regulation of apoptotic proteins and cell cycle-related regulatory proteins was also involved in the inhibition of tumor growth by piperine in vivo, the tumors were harvested from each group and the expression level of these proteins were analyzed by Western blot. As shown in Figure 6B, the level of active caspase 3 was increased and cyclin B1 was reduced in tumors from the mice received piperine therapy, which was similar to the effect of piperine in vitro. Histopathological analysis of the tumor tissues excised from the control mice showed groups of large, round and polygonal cells, with pleomorphic shapes, hyperchromatic nuclei and binucleation. Several degrees of cellular and nuclear pleomorphism were seen. In the tumors excised from the mice treated with piperine, extensive areas of coagulative necrosis were observed ( Figure 6C).
Piperine also inhibited the lung metastasis of 4T1 tumors 4T1 cells can metastasize to various organs, such as lung, as early as d 10 after inoculation of 1×10 5 4T1 tumor cells [17] . To study the potential inhibitory effect of piperine on metastasis, we used a colonogenic assay to evaluate the metastases in the lung. We detected less metastasis in the lungs of the mice that had been given 5 mg/kg piperine treatment compared to the lungs of the control mice (Figure 7). The results indicated that piperine not only suppressed the local primary tumor growth,

Discussion
Piperine is a potent anticancer compound that has been    npg demonstrated in various cell types. However, the antitumor activity and action mechanism of piperine on mammary cancer cells still remain unknown. Our results firstly indicated that piperine possessed significant cytoxicity against 4T1 cells both in vitro and in vivo. It was shown piperine induced the apoptosis of 4T1 cells in a dose-dependent manner. We investigated the effect of piperine on the expression levels of the Bcl-2 family, the pro-apoptotic Bax and the anti-apoptotic Bcl-2, which regulate mitochondrial apoptosis. When Bax is overexpressed in cells, apoptotic death in response to death signals is accelerated, while Bcl-2 is overexpressed, it heterodimerized with Bax and the cell death is repressed. Following the exposure to piperine, we found no change of Bax and Bcl-2 at protein level in the 4T1 cells, indicating that the apoptosis induced by piperine is not dependent of the Bcl-2 pathway. We found that the up-regulation of caspase 3 activity may lead to the apoptosis in 4T1 cells. Members of the cyclin family of proteins are key regulators of the cell cycle. Cyclins bind and activate members of the cyclin-dependent kinase (Cdk) family to control cell cycle progression. During the G 1 phase, cyclins D1, D2, and D3 form complexes with cdk4 or cdk6, and cyclin E with cdk2, to modulate the expression of proliferative genes. Cyclin A associates with cdk2 during the S phase, and with cdc2 (cdk1) at the S-G 2 boundary and into G 2 . Progression through G 2 , culminating in mitosis, further requires that cdc2 form complexes with cyclins B1 and B2 [18] . We found that piperine treatment down-regulated the expression of cyclin B1 in 4T1 cells with a dose-dependent manner, while there was no change of cyclin D1 expression after piperine treatment. It is consistent with the observation that the percentage of 4T1 cells in G 2 /M phase was increased after piperine treatment, while no significant change in G 0 /G 1 phase cell population.
The results of the present study provide the potential application of piperine in tumor therapy. Bhat BG et al reported the metabolism of piperine including absorption, tissue distribution and excretion of urinary conjugates in rats [19] . Upon administration of piperine to male albino rats by gavage or intraperitoneally, about 97% was absorbed irrespective of the mode of dosing. Examination of the passage of piperine through the gut indicated that the highest concentration in the stomach and small intestine was attained at about 6 h [19] .
The anticancer activity of piperine in vivo was probably partly due to its direct antiproliferative effect on 4T1 cells possibly by increasing the caspase 3 activity and down-regulated the expression of cyclin B1. In this study, we also provided the evidence that piperine can suppress the metastasis of 4T1 tumor in vitro and in vivo. It is well known the process of tumor cell metastasis requires the degradation of ECM molecules in the basement membrane, which is the largest barrier between cancer cells and the bloodstream. The key proteases that are involved in ECM degradation contain MMPs. Our results showed that Piperine significantly reduced the mRNA expression of MMP-9 and MMP-13. It had been reported that suppression of the expression of MMP-9 in tumor cells by piperine is through the inhibition of PKCα and ERK phosphorylation and reduction of NF-κB and AP-1 activation [11] . MMP-13 was shown to be expressed in more invasive breast carcinoma cells [20] , and some evidence demonstrated that increased tumor-derived MMP-13 expression independently predicts poor prognoses [21] . In breast cancer, MMP-9 seems to be expressed in cancer tissue. Malignant breast tumors have increased MMP-9 activity compared to the benign tumors [22] . Nevertheless, MMP-9 expression has also been described as a positive prognostic marker in node-negative breast cancer [23] . In another study, positive stromal MMP-9 expression predicts poor survival in the hormone-responsive small tumors, whereas MMP-9 expression in carcinoma cells favors survival [24] . Thus, MMP-9 expression is associated with both inhibition and stimulation of tumor growth and progression. So, the exact mechanism of the inhibitory effect of piperine on tumor metastasis in vivo needs to be further elucidated. It have been supposed that the inhibition effects of piperine on oxygenase, p450 isoenzyme and cycloxygenase-1 expression may contribute to the antimetastatic qualities [25] .
According to some authors, the antitumor activity of piperine may be related to its immunomodulatory properties, which involves the activation of cellular and humoral immune responses [26,27] . It was proved to be a potent inhibitor of NF-κB, c-Fos, CREB, ATF-2, and proinflammatory cytokine gene expression in B16F-10 melanoma cells [10] . Other researches documented the chemopreventive efficacy of Piperine is probably due to their antilipidperoxidative and antioxidant potential as well as its modulating effect on the carcinogen detoxification process [28,29] . Piperine is also known to enhance the bioavailability of some drugs by inhibiting drug metabolism or by increasing absorption [12,30,31] . Piperine with other antitumor drugs such as 5-FU [12] , piplartine [13] augmented the antitumor activity and also decreased the side effects.
In conclusion, piperine can inhibit tumor growth by inducing cell apoptosis and cell cycle blockage. Also it can suppress 4T1 tumor growth and metastasis in vivo. Further studies will need to be carried out to determine the exact mechanisms of the antitumor action of piperine, and to investigate how it can