Selectivity of Pinus sylvestris extract and essential oil to estrogen-insensitive breast cancer cells Pinus sylvestris against cancer cells.

BACKGROUND: So far, the anticancer action of pine tree extracts has mainly been shown for the species distributed widely around the Asian countries.
OBJECTIVE: Therefore, this study was performed to examine the potential cytotoxicity of Scots pine (Pinus sylvestris L.) native also to the European region and growing widely in Estonia.
MATERIALS AND METHODS: The cytotoxic activity of methanol extract and essential oil of Scots pine needles was determined by sulforhodamine B assay in different human cancer cell lines.
RESULTS: This needle extract was found to suppress the viability of several human cancer cell lines showing some selectivity to estrogen receptor negative breast cancer cells, MDA-MB-231(half maximal inhibitory concentration [IC50] 35 μg/ml) in comparison with estrogen receptor-positive breast cancer cells, MCF-7 (IC50 86 μg/ml). It is the strongest cytotoxic effect at all measured, thus far for the needles and leaves extracts derived from various pine species, and is also the first study comparing the anticancer effects of pine tree extracts on molecularly different human breast cancer cells. The essential oil showed the stronger cytotoxic effect to both negative and positive breast cancer cell lines (both IC50 29 μg/ml) than pine extract (IC50 42 and 80 μg/ml, respectively).
CONCLUSION: The data from this report indicate that Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment.

INTRODUCTION

Breast cancer affects more than 1.3 million women worldwide each year and accounts for about 14% of cancer-related deaths.[12] In western countries, the woman's lifetime risk of developing this disease is more than 10%.[3] The incidence has increased in the past decades and is expected to rise substantially in the coming years.[2] Breast cancer is a heterogeneous group of pathologic entities consisting of several molecular subtypes, each with distinct natural histories and biological behaviors requiring also different management approaches.[24] Most newly diagnosed breast carcinomas (about 70%) are estrogen-receptor (ER)-positive and can be classified as luminal subtypes. Determining the ER status of tumor samples is a standard practice in clinical oncology as the patients with ER-positive tumors have likely benefit from endocrine therapies, and have generally a better prognosis than those with ER-negative breast cancers for which the therapeutic options are more complicatedand prognosis is worse.[125]

In recent years, interest in natural plant components with potential anticancer effects has continuously grown and considerable attention has focused on identifying chemopreventive phytochemicals to slow, reverse or completely halt the multistage process of carcinogenesis.[678910] Plants provide an extensive reservoir of natural products with a wide structural diversity and offer novel and exciting chemical entities in modern medicine.[11] The majority of current antitumor drugs have severe side effects accompanying their therapeutic action and, therefore, studies of traditional herbs and identifying novel natural products with high anticancer activity, but low cytotoxicity in normal cells are receiving considerable attention in the field of anticancer studies.[101112]

Pine trees are widely distributed worldwide being with around 105 species the largest genus of conifers.[131415] Consumption of various parts, including needles, bark, cones, and pollen is believed to promote health and prevent some aging-related chronic diseases.[13] There is growing evidence that pine needles can exert also antioxidant, antimutagenic, and antiproliferative effects on cancer cells.[8] Bark has been used in traditional medicine for over 2000 years as a nutritional supplement and phytochemical remedy.[16] Extracts derived from the bark of Pinus maritima Lam. (Pycnogenol and Flavangenol) and Pinus massoniana Lamb. may hold promise as anticancer agents and are good candidates for chemoprevention or chemotherapeutics in the future.[616] They can strongly inhibit the migration capability of human cervical cancer HeLa cells and induce selectively, the apoptosis of human liver cancer Bel-7402 and HepG2 cells.[910121617] Pycnogenol has been shown to exert antileukemic effects and protective properties against skin carcinogenesis.[671819] Moreover, it can selectively induce cell death in human mammary cancer MCF-7 cells, but not in normal human mammary MCF-10 cells.[1920] However, extracts prepared from needles of different pine species (Pinus thunbergii Parl., Pinus rigida Mill., Pinus koraiensis Siebold and Zucc., Pinus densiflora Siebold et Zuccarini) reveal only very limited anticancer effects on breast adenocarcinoma MCF-7 cells with half maximal inhibitory concentration (IC50) values in the range of more than 200 μg/ml.[1321]

There are no reports available about the potential anticancer action of extracts from Scots pine (Pinus sylvestris L.) growing natively in Europe and Asia and being a very common coniferous tree in Estonia.[22] For this reason, we evaluated the effects prepared from needles of P. sylvestris on various human cancer cell lines, and performed for the first time the comparative analysis of action of pine extract on estrogen receptor positive and negative breast carcinoma cells.

MATERIALS AND METHODS

Cell culture

Human cancer cell lines including ER-positive breast cancer MCF-7, ER-negative breast cancer MDA-MB-231, prostate cancer LNCaP, gastric carcinoma MKN7, colon adenocarcinoma SW480, oral epidermoid carcinoma KB, lung adenocarcinoma LU-1, liver hepatocellular carcinoma HepG2, and promyelocytic leukemia HL-60 cells were cultured in Dulbecco's Modified Eagle Medium or RPMI-1640 cell culture medium, both supplemented with 10% fetal bovine serum. Cells were cultivated at 37°C in a humidified atmosphere containing 5% carbon dioxide.

Plant material and preparation of extracts

Pine needles collected from Northern Estonia were dried and crushed to a fine powder, and then extracted with methanol for three times (48 h per time) at room temperature (20°C). Next, the methanol extracts were recovered under reduced pressure to obtain crude extracts, which were used in the cytotoxic assays. Extracts were dissolved in dimethyl sulfoxide (DMSO) to prepare 4 mg/ml stock solutions that were later mixed with the cell culture medium to achieve the desired concentrations. The final test concentrations were 0.8, 4, 20, and 100 μg/ml.

In vitro cytotoxic assay

The effects of pine needle extracts on the viability of malignant cells were determined by sulforhodamine B cytotoxic assay.[23] Briefly, cells were grown in 96-well microtiter plates with each well containing 190 μl medium. After 24 h, 10 μl of test samples dissolved in DMSO were added to each well. One plate with no samples served as a day 0 control. The cells were continuously cultured for additional 48 h, fixed with trichloroacetic acid and stained with sulforhodamine B, followed by the determination of optical densities at 515 nm using a Microplate Reader (BioRad). The percentage of growth inhibition was calculated using the following equation:

Where, OD is optical density or absorbance values. The potent anticancer agent ellipticine and tamoxifen citrate were used as a positive control.

Isolation of essential oil

The essential oil was isolated from fresh pine needles by the hydrodistillation method described in a previous study.[24] The pine oil used for the cytotoxic assay was also hydrodistilled.

Gas chromatography–mass spectrometry analysis

Gas chromatography-mass spectrometry (GC-MS) analysis was carried out using an Agilent 5975 Series Mass Selective Detectors, Agilent 7890A GC (Agilent Technologies, Inc.) with two detectors (MS and FID) on a fused silica capillary column (30 m × 0.25 mm) with a bonded stationary phase: Poly (5%-diphenyl-95%-dimethyl) siloxane (DB-5). The film thickness of the stationary phase was 0.25 mm. The carrier gas was helium with the split ratio of 1:30 and the flow rate of 1.3 ml/min was applied. The temperature program was from 50°C to 240°C at 2°C/min; the injector temperature was 300°C. The MS detector was operated in the EI mode of 70 eV, and at a scan rate of 2 scans/s with an acquisition mass range of 20–400 u.

Statistical analysis

Cytotoxic data were calculated and expressed as concentrations, at which 50% of cell growth was inhibited (IC50 values ± standard deviation). All experiments were carried out in triplicate and the Table Curve 2Dv4 software was used for calculation of IC50 values. P < 0.01 were considered significant.

RESULTS AND DISCUSSION

A dose-dependent decrease in viability of human breast cancer cells was observed after 48 h of treatment with 0.8, 4, 20, and 100 μg/ml of pine needles extract. The effect on ER-negative MDA-MB-231 cells was almost three-fold stronger (IC50 35.56 μg/ml) than for ER-positive MCF-7 cells (IC50 86.37 μg/ml) indicating some selectivity of pine needles extract to hormone refractory breast cancer cells [Figure 1].

Figure 1

Cytotoxic effect of Scots pine needles extract on human breast cancer cell lines sulforhodamine B assay

The half-maximal cytotoxic effects of methanol extract prepared from Scots pine needles on other malignant cell lines studied in this work remained in the concentration range of 50 μg/ml to 80 μg/ml [Table 1], being somewhat stronger on leukemia, colon cancer, lung adenocarcinoma, and hepatocellular carcinoma cell lines compared to oral epidermoid carcinoma KB cells.

Table 1

Cytotoxic activity of extract prepared from Scots pine (Pinus sylvestris L.) needles on various cancer cell lines measured by sulforhodamine B assay

Previously, extracts prepared from needles of various pine species have been shown to exert some anticancer effects. However, cytotoxicity expressed to human breast cancer cells has described only at very high concentrations (IC50 values certainly more than 200 μg/ml) and for species growing mainly in the Asian region.[1321] In this work, we demonstrated for the first time that methanol extract derived from needles of Scots pine tree widely and natively distributed throughout Estonia has potential to suppress the viability of human breast cancer MCF-7 cells. These cells are derived from a patient with metastatic breast cancer, and earlier studies have shown that estrogen can directly stimulate the growth of this tumor cell line.[20] Moreover, the same extract revealed almost three-fold stronger cytotoxic activity on MDA-MB-231 human breast cancer cells, which do not express estrogen receptor-a and are, therefore, not responsive to endocrine treatment. These data indicate that Scots pine needles can contain some compounds with the high potential to be developed as candidates for chemoprevention or chemotherapeutic adjuvants for endocrine insensitive breast tumors.

Phytochemical analyses of pine needles have found numerous compounds as possible effective components.[1315] Needles are especially rich in various polyphenols, which may exert different beneficial effects on human health.[1325] Indeed, three structurally related 6-C-methyl flavonoids were recently isolated from twigs and leaves of Pinus densata Mast. showing some antiproliferative activity also on ER-positive human breast cancer ZR-75-30 cells.[26] Polyphenolic compounds are also major ingredients of pine bark extracts that contain more than 40 kinds of natural bioactive components among, which procyanidins are the major substances.[2728] Pycnogenol as an extract from French maritime pine bark is a mixture composing mainly of procyanidins, flavonoids, and phenolic acids.[671920] This extract lacks toxicity, is nonteratogenic and non-mutagenic, and can selectively induce cell death in human mammary cancer MCF-7 cells, but not in normal human mammary MCF-10 cells.[910121920] However, proanthocyanidins from bark extract of P. massoniana Lamb expressed only very weak inhibitory effect on the growth of human mammary cancer MDA-MB-231 cells with IC50 value essentially higher than 200 μg/ml.[29] Taken together, this can indicate that either the profile of specific polyphenolic compounds with anticancer activity varies in distinct parts of pine tree being somewhat different in needles and bark; various pine species contain different bioactive compounds; or there are some nonphenolic substances responsive for the antiproliferative effect of P. sylvestris needle extract on ER-negative breast cancer cells.

In addition, the cytotoxic activity of both Scots pine extract and essential oil was compared by us on ER-positive MCF7 and ER-negative MDA-MB-231 using also tamoxifen citrate and ellipticine as a positive control [Table 2]. The data revealed that the Scots pine essential oil exhibited stronger cytotoxic effects on both breast cancer cell lines than the extract: IC50 values of oil were 28.67 and 29.23 μg/ml, respectively, but 80.20 and 42.27 μg/ml for the extract studied. Thus, the cytotoxic activity of the essential oil was practically the same on both cell lines.

Table 2

Cytotoxic activity of Scots pine needles extract and essential oil on MCF-7 and MDA-MB-231

It was only very recently demonstrated that essential oils from the needle extract of Pinus roxburghii Sarg. express a rather strong (about 70%) cytotoxic activity on human breast cancer MCF-7 cells at the dose of 100 μg/ml. This effect was likely related to the high concentrations of terpinen-4-ol, (E)-caryophyllene, and a-humulene in the needle essential oil.[15] However, the content of these substances in the essential oil hydrodistilled from fresh Scots pine needles of Estonian origin is only very small (0.1% to 0.3%), and the principal constituents were a-pinene (48.1%) and camphene (10.1) [Table 3]. Low concentrations of terpinen-4-ol and a-humulene (0.05–0.4%), as well as somewhat higher amount of (E)-caryophyllene (2.9%) has been determined in Estonian pine oil also previously.[30] It is interesting to compare that the essential oil of juniper (Juniperus communis L.) growing in Estonia contained at the mean 0.4% of terpinen-4-ol, 1.1% of (E)-caryophyllene, and 0.9% of α-humulene.[24]

Table 3

Composition of the essential oil hydrodistilled from needles of Pinus sylvestris

This report describes for the first time the anticancer effects of needle extracts and essential oil obtained from P. sylvestris on various human malignant cell lines showing a clear cytotoxic selectivity to ER-negative breast cancer cells compared to ER-positive cell line. Although, the exact isolation and structure elucidation of the active compound(s) is needed to perform in the further studies it is likely that Scots pine needle extract and oil may serve as an easily accessible source of potential candidate for the development of novel therapeutic anticancer agents.

CONCLUSION

The Scots pine needles extract and essential oil exhibits some potential as chemopreventive or chemotherapeutic agent for mammary tumors unresponsive to endocrine treatment.