Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells

Background Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves), a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Methods Hydro-methanolic extract of curry leaves (CLE) was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau’s method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. Results CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Conclusions Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification of active component(s) from the leaf extract could lead to the development of anti-cancer agents which could be useful in the treatment of different types of cancers.


Background
Breast cancer is the second most prevalent cancer in the world next only to lung cancer [1] and is a major public health problem in developing countries like India. Every year 75,000 new cases of breast cancer are reported in India. The increase in the number of cases has been attributed to factors such as genetics, environmental pollution, urbanization and changing food habits.
Murraya koenigii Spreng (curry-leaf tree), is a small aromatic tree belonging to the family Rutaceae. It is a tropical to sub-tropical tree native to India. Of the 14 global species belonging to the genus Murraya, only two are available in India, namely, M. koenigii and M. paniculata. Of the two M. koenigii is more popular due to its large spectrum of medicinal properties. M. koenigii leaves have a slightly pungent, bitter and feebly acidulous taste and these characteristics are retained even after drying. Fresh and dried curry leaves are extensively used in South Indian culinary practices for seasoning and flavouring dishes [2]. Different parts of the plant such as leaves, root, bark and fruit are known to possess various biological activities. Traditionally, this plant is used in Indian systems of medicine for a variety of ailments and also used as a tonic, stomachic, and carminative [3][4][5].
We and others have shown that a hydro-methanolic extract of M. koenigii leaves is rich in phenolic content [10,21]. Polyphenols have a wide spectrum of biological activities, including anti-oxidant, anti-inflammatory and metal-chelating properties [22,23]. Recent studies have shown that naturally occurring polyphenols/flavanoids modulate the functionality of the 26S proteasome, a multienzymatic, multi-catalytic complex localized both in the cytoplasm and nucleus of eukaryotic cells [24,25]. The 26S proteasome is a huge 2.4 MDa complex comprising of two sub-complexesthe 19S regulatory subunit and the 20S catalytic subunit [26]. The 20S sub-unit possesses at least three distinct activities, which are associated with the three different β subunits respectively: chymotrypsin-like activity (β5), trypsin-like activity (β2) and the caspase-like activity (β1) [27]. The 26S proteasome is the major non-lysosomal pathway of protein degradation in eukaryotic cells. This proteolytic machine is involved in the degradation of oxidized, unfolded and misfolded proteins and antigen presentation [28][29][30][31]. It regulates several cellular processes such as apoptosis, signal transduction, cell-cycle regulation and cell differentiation [32]. Two important functions of the proteasome system are to promote tumor cell proliferation and protect tumor cells against apoptosis [27,33,34].
In the present work, we demonstrate for the first time that the hydro-methanolic extract of M. koenigii leaves rich in phenolic content, potently inhibits the activity of the proteasome both in vitro and in vivo. The CLE induced cell death in two breast cancer cell lines in a time and dosedependent manner. The leaf extract altered the growth kinetics of the cancer cells in a dose-dependent manner as demonstrated by the colony formation assay. Cancer cells but not normal cells were arrested in the S phase of the cell cycle. Annexin V binding experiments demonstrate that apoptosis was induced by CLE in both the breast carcinoma cell lines.

Preparation of curry leaf extracts
Curry leaves were collected from the local area from a single tree. Identity of the curry leaves was confirmed by Dr. B. Pratibha Devi, Professor and Head, Department of Botany, Osmania University, Hyderabad, India. A voucher specimen (voucher no: 068) was deposited in a herbarium at the Department of Botany, Osmania University, Hyderabad, India. The leaves were washed and air dried in shade for 3 weeks. After drying, the leaves were ground to a fine powder using an electric mixer grinder. The leaf powder was extracted with 80% methanol in water by keeping on a vortex mixer for 3-4days. This was followed by centrifugation of the extract at 5000 rpm for 30 min. The supernatant was filtered using a 0.4 μm filter (Millipore). The resultant Methanol: Water extract was stored at −20°C and was used for all our studies. These extracts designated as 'CLE' , were used in the cell-culture assays at different doses based on their total phenolic content [equivalent to μg of gallic acid (GAE)] measured spectrophotometrically by the Folin-Ciocalteau method.

Total Phenolic Content (TPC)
The total phenolic content of the extract was determined with Folin-Ciocalteau's reagent using Gallic acid as a standard [35]. Different concentrations of Gallic acid standards (20-100 μg/μl) and CLE samples were taken in glass test tubes and volume was made up to 150 μl with distilled water. 750 μl of 10% Folin's reagent was added and kept for 5 minutes at room temperature, followed by addition of 750 μl of 6% Na 2 CO 3 and vortexed for 5 minutes. The tubes were then incubated for 90 minutes at room temperature. The absorbance was measured at 725 nm in a Hitachi double beam spectrophotometer. The final concentration of the total polyphenols present in the extract was expressed as μg of Gallic Acid Equivalents (GAEs).

MCF-7 and MDA-MB-231 (breast carcinoma cell lines)
and WI-38 (normal human lung fibroblasts) were obtained from the National Centre for Cell Sciences, Pune, India. Cells were maintained in DMEM supplemented with 10% FBS, 2 mM Higluta-XL, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.5 ng/ml amphotericin B, 1 mM sodium pyruvate and 1X non-essential amino acid mixture. Cells were maintained and grown in a humidified atmosphere at 37°C and 5% CO 2 . WI-38 cells were grown for no more than 30 passages, as recommended by European Collection of Cell Cultures (ECACC).

Cell viability/proliferation assay
Cell viability was determined by quantification of 3-(4,5dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases. In brief, 1 × 10 5 cells/well were plated in a 96-well plate and incubated with different concentrations of the CLE for either 12 h or 24 h or with MG-132 for 24 h. Following this, MTT was added to a final concentration of 100 μg/well and further incubated for 3 h at 37°C. The formazan dye crystals formed were solubilized in DMSO and the plate was incubated at room temperature for 1 h. The absorbance was measured at 595 nm in an ELISA microplate reader (Biotek, New York, USA). All samples were assayed in triplicate in three independent experiments. Absorbance values plotted are the mean from three independent experiments and the results are expressed as percentage of the control, which was considered to be 100%.
Colony formation assay MCF-7 and MDA-MB-231 cells were plated in duplicate at a density of 1 × 10 4 and 1 × 10 3 cells/ml respectively in 6-well plates. Next day, the cells were treated with varying concentrations of CLE. Plates were incubated at 37°C and 5% CO 2 for one week. After a week, the colonies were fixed with 4% formaldehyde for 15mins followed by staining with 0.005% crystal violet. The colonies were photographed with a digital Nikon D90 camera. Three independent experiments were done with each cell line.

Cell cycle analysis and annexin V binding assay
Cell cycle analysis: MCF-7 or MDA-MB-231 or WI-38 cells were plated at a density of 1.5 × 10 6 cells in a 10 cm dish. Next day, cells were treated with different doses of the CLE. After 24 h incubation, cells were harvested and suspended in 1X-PBS containing 2% FBS. The cells were fixed with 70% ethanol at 4°C for 1 h followed by the addition of propidium iodide (5 μg/ μl) and RNase (10 μg/ μl) and further incubated for 3 h at 4°C. The DNA content was evaluated in a flow cytometer (BD FACS ARIAII,  USA). The data was analyzed using Modfit software (BD Biosciences, USA). Annexin-V staining: MCF-7 or MDA-MB-231 cells were plated at a density of 1.5 × 10 6 cells in a 10 cm dish. Next day, cells were treated with different doses of the CLE. After 24 h incubation, cells were washed with 1X-PBS and re-suspended in 100μl binding buffer (supplied by the vendor). Cells were stained with Annexin V-FITC and propidium iodide according to the manufacturer's protocol before analysis by flow cytometry.

Inhibition of purified 20S proteasome activity
Chymotrypsin-like activity of the purified 20S proteasome was measured as follows: In brief, 200 ng of purified 20S proteasome was incubated in 200μl of assay buffer (50 mmol/L Tris-HCl, pH 8.0 containing 0.035% SDS) with or without different concentrations of CLE and 40 μM substrate Suc-Leu-Leu-Val-Tyr-AMC (for chymotrypsinlike activity) and incubated for 2h at 37°C. The free 7amino-4-methylcoumarin (AMC) liberated was measured fluorimetrically using a multi-mode reader [Spectra Max M5] using an excitation filter (380 nm) and emission filter (460 nm). The data is plotted as mean (+/− standard error) and is expressed as a percentage of the control, which was considered to be 100%. The assay was repeated thrice.

Inhibition of 26S proteasome activity in intact cells
To measure inhibition of the proteasome activity in living tumor cells, MCF-7 or MDA-MB-231 or WI-38 cells were plated at a density of 1 × 10 4 in a 24 well plate. Next day, cells were treated with or without the CLE at the indicated concentrations. After 24 h of treatment, the media was aspirated out and 500 μl of 1X-PBS was added followed by addition of fluorogenic substrates (20 μM final concentration) specific for the chymotrypsinlike (Ch-L), trypsin-like (T-L) and caspase-like (Cp-L) activities of the 20S proteasome. The plate was then incubated for 2 h at 37°C. 200 μl of the 1X-PBS was then transferred into a black plate and the free 7-amino-4-methylcoumarin (AMC) liberated was measured fluorimetrically in a multi-mode reader [Spectra Max M5] at excitation (380 nm) and emission (460 nm). The results are displayed as mean (+/− standard error) and are expressed as a percentage of the control, which was considered to be 100%. All samples were assayed in triplicate in three independent experiments.

Inhibition of 26S proteasome activity in cell extracts
MCF-7 or MDA-MB-231 cells (1 × 10 7 ) were harvested, washed twice in 1X-PBS and re-suspended in 1 ml ATP/ DTT lysis buffer [10 mmol/L Tris-HCl (pH 7.8), 5 mmol/ L ATP, 0.5 mmol/l DTT, 5 mmol/L MgCl 2 ]. Cells were incubated on ice for 10 minutes, followed by sonication for 15 seconds. The lysate was centrifuged at 2000 rpm for 10 minutes at 4°C. The supernatant enriched in 26S proteasomes is the cell extract which was used for the assay. This was mixed with glycerol (20% final concentration), aliquoted and stored at −80°C, and was stable for at least 1 month. The total protein content of the cell extract was estimated by the Bicinchoninic Acid (BCA) method using a kit (Bangalore Genei, Bangalore, India). The assay was carried out in a total of 200 μl reaction volume containing proteasome extract (50 μg protein), 50 mM EDTA, varying concentrations of the CLE/MG-132 and 50 μM of the proteasomal fluorogenic substrates and incubated for 2 h at 37°C. The amount of free 7-amino-4-methylcoumarin (AMC) liberated was measured fluorimetrically. The results are expressed as mean (+/− standard error) as a percentage of the control, which was considered to be 100%. All samples were assayed in triplicate in three independent experiments.

Statistical analysis
All experiments were performed in triplicates and repeated at least three times and the data are presented as mean+/− SEM. Mean values were compared across concentrations of CLE using non-parametric test of Kruskal-Wallis one-way ANOVA for each cell line using the SPSS statistical software. Differences between groups were considered significant at α (probability) level of </= 0.05.

M. koenigii leaf extract alters viability and growth kinetics of breast cancer cells
The TPC of the methanolic extract of curry leaves was 3 μg of GAEs/μl of the CLE. MTT assays were performed with different concentrations of CLE (GAE) in both the cell lines at the 12 h and 24 h time points to assess the effect of the extract on cell viability. There was a significant (p<0.05) time and dose-dependent decrease in cell viability in both the cell lines. As expected, the decrease in cell viability observed after 24 h of treatment was higher compared to 12 h. In addition, MDA-MB-231 cells ( Figure 1A and 1B) appeared to be more sensitive to CLE induced cell death than MCF-7 cells (Figure 1D and 1E). 24 h post treatment, a 50% reduction in cell viability was observed in MDA-MB-231 and MCF-7 cells at 15 μg and 37.5 μg GAE of CLE respectively. As a positive control, the effect of MG-132-a specific inhibitor of the proteolytic activity of the 26S proteasome was also assessed on cell viability in both the breast cancer cell lines. 24 h post treatment with MG-132, a 50%    than MCF-7 cells to the CLE induced effects. Interestingly, CLE had no effect on cell cycle in the normal WI-38 cell line at any of the concentrations tested ( Figure 5 and Table 3), indicating that CLE could arrest growth only in cancer but not normal cells.
Further, Annexin-V binding experiments were conducted in both cell lines to determine the probable mechanism of cell death. That 6μg CLE resulted in 45% of live MDA-MB-231 cells whereas, a dose of 12.5 μg CLE was required for a similar effect in MCF-7 cells not only confirms the greater sensitivity of MDA-MB-231 than MCF-7 cells but also suggests apoptosis to be the probable mechanism of cell death. This is confirmed by our finding that CLE demonstrated a dose dependent increase in the % of apoptotic cells in both MDA-MB-231 and MCF-7 cells. 50% of cells were apoptotic with 6 μg CLE in MDA-MB-231 cell line and it increased to 66% at a dose of 37.5 μg CLE ( Figure 6). A similar dose dependent increase in the % apoptotic cells was seen in MCF-7 cells (Figure 7).

M. koenigii leaf extract inhibits 20S purified proteasome activity
We then tested whether or not CLE inhibited the activity of the purified 20S rabbit proteasome in a cell-free system. Indeed CLE decreased the chymotrypsin-like activity of the 20S proteasome in a dose-dependent manner and a 50% decrease (IC 50 ) in activity was seen at a concentration of CLE equivalent to 3 ug of Gallic Acid (GAE)/μl of the extract (Figure 8).

M. koenigii leaf extract inhibits cellular 26S proteasome activity in intact cells
Whether the CLE also inhibited the activity of the 26S proteasome in living cancer cells was assessed next in both MCF-7 and MDA-MB-231 cells. Similar to its effects on the purified 20S rabbit proteasome, CLE showed a significant (p<0.05), dose-dependent decrease in the chymotrypsin-like, trypsin-like and caspase-like activities of the 26S proteasome in intact cancer cells (Figures 9 and 10).
On the other hand, CLE did not inhibit the chymotrypsin-like activity of the 26S proteasome at any of the concentrations tested in the normal WI-38 cells ( Figure 10D) indicating the specificity of the effect to cancer cells.  (Figures 11 and 12). As a positive control MG-132, a proteasome inhibitor was also tested. It was observed that MG-132 decreased the chymotrypsinlike activity of the 26S proteasome in a dose-dependent manner in cell extracts prepared from both MDA-MB-231 and MCF-7 cells with an IC 50 of >50 nM and 25 nM in the two cell lines respectively (Figures 11D & 12C).

Discussion
Search for new anti-cancer drugs from natural sources is one of the most important approaches for cancer prevention and treatment. In recent years, more emphasis is laid on Complementary and Alternative [CAM] forms of medicine for the treatment of various cancers, among which herbal medicine is now being explored for cancer therapy [36]. Dietary constituents have chemopreventive and chemotherapeutic potential, in addition to ameliorating the side effects associated with conventional chemotherapy. In this context, a recent approach in cancer therapy advocates the inhibition of the proteolytic activity of 26S proteasome, the multi-enzymatic protease complex in cells. Unlike normal cells, cancer cells have increased proteasomal activity which is essential for their survival and uninhibited proliferation [37][38][39]. Inhibition of the proteasome results in apoptosis and cancer cell death [40]. Importantly, inhibitors of the 20S proteolytic unit of the proteasome have been shown to induce apoptosis and cell cycle arrest only in neoplastic cells but not in normal cells [39][40][41]. Therefore, the proteasome has emerged as an attractive molecular target for cancer therapy [42]. A number of synthetic proteasome inhibitors have been described and most of them interfere with the proteolytic activity of the β subunits of the 20S proteasome. These inhibitors which bind the active site either reversibly or irreversibly include peptide aldehydes such as MG-132, non-peptide inhibitors such as lactacystin and epoxomycin and peptide boronates such as bortezomib [43]. Bortezomib/Velcade/PS-341 is the first-in-line and the only dipeptide boronate proteasome inhibitor to be approved by the FDA in 2003 for the treatment of patients with refractory multiple myeloma. Bortezomib is now being tested in a variety of hematological and solid tumors including non-Hodgkin's lymphoma, prostate, breast and non-small-cell-lung cancer [44,45]. In recent years, synthetic polyphenols such as apigenin, epigallocatechin gallate [EGCG], quercitin and myrcetin have been reported to act as proteasome inhibitors and induce cell death in cancer cells [24]. Drug resistance limits the effectiveness of existing treatment options and is a major challenge faced in current cancer research. Interestingly, it has been shown that lactacystin and bortezomib enhance sensitivity of cancer cells that are resistant to routine chemotherapy [46,47]. Nevertheless, synthetic proteasome inhibitors are associated with some toxicity. Therefore, proteasome inhibitors from natural food sources with minimal or no toxicity can be potential anticancer agents.
In the present study, we report the anticancer potential of M. koenigii leaf extracts in two human breast carcinoma cell lines. In recent years, dietary polyphenols have attracted lot of attention owing to their anti-tumor activities [48,49]. One such activity is the inhibition of the proteasome in cancer cells leading to cell death. Recent work from our laboratory [21] has demonstrated that M. koenigii leaf extract is a rich source of polyphenols. In this study, we found that a hydro-methanolic extract of curry leaves is rich in polyphenol content. Extracts of M. koenigii leaves have been reported to possess various biological activities such as anti-diabetic, anti-oxidative and anti-inflammatory [8][9][10]. Recently, carbazole alkaloids from M. koenigii have shown anti-cancer activity in leukemia cells [18][19][20]. However, the underlying mechanism(s) are not reported yet. In the present work, we demonstrate for the first time that the hydro-methanolic extract of curry leaf has proteasome-inhibitory potential and induces cell death in human breast cancer cells.
We found that the methanolic extract of curry leaves significantly decreased cell viability and proliferation of both MCF-7 and MDA-MB-231 breast cancer cells in a dose-dependent manner. This was further supported by the significant reduction in the number of colonies in CLE treated cells compared to vehicle treated cells. Our cell viability and colony formation data shows that CLE altered the growth kinetics of both MCF-7 and MDA-MB-231 cells. Therefore, curry leaves appear to be a promising drug candidate for restricting the growth of breast cancer cells.
In order to assess the stage at which the cell growth was arrested by CLE, we performed cell cycle experiments and observed that there was a clear arrest of cells in the synthetic or S phase. In contrast to its effect on the breast carcinoma cell lines, CLE interestingly, had no effect on the different phases of the cell cycle in the normal fibroblast cell line. Anti-cancer drugs can result either in programmed cell death/apoptosis or necrosis. In order to identify the probable cell death pathway involved, we used Annexin V binding to test if the cell death occurred through apoptosis or necrosis. Indeed it was found that in both the breast cancer cell lines CLE induced apoptotic cell death.
We tested next, whether the anti-cancerous effect of the CLE was due its potential of inhibiting the proteolytic activity of the protein degrading machine present in eukaryotic cellsthe 26S proteasome, which is now considered to be a novel approach for cancer therapy. We observed that CLE inhibited the purified 20S proteasome enzyme. Furthermore, it significantly inhibited all the three enzymatic activities associated with the 26S proteasome in living cells in a dose-dependent manner. To further confirm the findings in live cells, cell extracts were prepared from both breast cancer cells and tested for the potential of the CLE in inhibiting the cellular proteasome. Similar to our findings in intact cells we found that CLE inhibited the 26S proteasome in cell extracts also in a dose-dependent manner. To test whether or not the inhibitory effect of the CLE on the proteasome activity was specific to cancer cells, we tested its effects in WI-38, a normal lung fibroblast cell line. Interestingly, CLE had no effect on the chymotrypsin-like activity of the 26S proteasome in live WI-38 cells. Hence, data from our proteasome-inhibition experiments suggests that the CLE could inhibit the cellular proteasome leading to cell death in cancer cells but not normal cells. Our data is in accordance with earlier reports that proteasome inhibitors selectively inhibited proteasome activity only in neoplastic cells [39,50,51].

Conclusion
Our results indicate that the hydro-methanolic extract of curry leaves is a good source of active compound(s) that can potentially inhibit the 26S proteasome specifically in cancer cells. The inhibition of the proteasome in cancer cells appears to be one of the important biological activities in M. koenigii leaves that can be exploited for cancer treatment. Hence, isolation and characterization of active component(s) from methanolic extracts of curry leaves could lead to the discovery of novel anticancer agents.