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. 2012 Dec 13;12:253.
doi: 10.1186/1472-6882-12-253.

Frankincense Essential Oil Prepared From Hydrodistillation of Boswellia Sacra Gum Resins Induces Human Pancreatic Cancer Cell Death in Cultures and in a Xenograft Murine Model

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Frankincense Essential Oil Prepared From Hydrodistillation of Boswellia Sacra Gum Resins Induces Human Pancreatic Cancer Cell Death in Cultures and in a Xenograft Murine Model

Xiao Ni et al. BMC Complement Altern Med. .
Free PMC article

Abstract

Background: Regardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model.

Methods: Boswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0-2 h, Fraction II at 8-10 h, and Fraction III at 11-12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11-12 h (Fraction IV). Chemical compositions were identified by gas chromatography-mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry.

Results: Longer duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic acids, in frankincense essential oil fraactions. Human pancreatic cancer cells were sensitive to Fractions III and IV (containing higher molecular weight compounds) treatment with suppressed cell viability and increased cell death. Essential oil activated the caspase-dependent apoptotic pathway, induced a rapid and transient activation of Akt and Erk1/2, and suppressed levels of cyclin D1 cdk4 expression in cultured pancreatic cancer cells. In addition, Boswellia sacra essential oil Fraction IV exhibited anti-proliferative and pro-apoptotic activities against pancreatic tumors in the heterotopic xenograft mouse model.

Conclusion: All fractions of frankincense essential oil from Boswellia sacra are capable of suppressing viability and inducing apoptosis of a panel of human pancreatic cancer cell lines. Potency of essential oil-suppressed tumor cell viability may be associated with the greater abundance of high molecular weight compounds in Fractions III and IV. Although chemical component(s) responsible for tumor cell cytotoxicity remains undefined, crude essential oil prepared from hydrodistillation of Boswellia sacra gum resins might be a useful alternative therapeutic agent for treating patients with pancreatic adenocarcinoma, an aggressive cancer with poor prognosis.

Figures

Figure 1
Figure 1
Suppression of pancreatic cancer cell viability in response to frankincense essential oil treatment. Human pancreatic cancer cells (5 × 103 in 100 μl) were seeded in each well of 96-well tissue culture plates for adherence. Cells were subjected to serial dilutions of frankincense essential oils treatment, (A) Fraction I obtained at 78°C hydrodistillation for 0–2 h, (B) Fraction II obtained at 78°C for 8–10 h. (C) Fraction III obtained at 78°C for 11–12 h, or (D) Fraction IV obtained at 100°C for 11–12 h. All experiments were prepared in triplicate. Cell viability was determined at 24 h following essential oil treatment using the XTT colorimetric assay. Results are presented as mean cell viability (%) ± standard error of mean (SEM) in relation to the number of cells at the time of essential oil treatment from at least 4 independent experiments. * indicates statistical difference in cell viability between essential oil-treated and untreated cells (P < 0.05).
Figure 2
Figure 2
Quantitative analysis of frankincense essential oil-induced pancreatic cancer cell death. Human pancreatic cancer cells were seeded into each well of 96-well tissue culture plates at the concentration of 5 × 103/100 μl. After adherence, cells were subjected to serial dilutions of either (A) Fraction III or (B) Fraction IV essential oil treatment. At 3 h post-treatment, cell viability was determined by the LDH cytotoxicity detection kit. Experiments were prepared in triplicate; and results are presented as mean % of cell death ± SEM in relation to untreated cells from at least 3 independent experiments. * indicates statistical difference of cell death between essential oil-treated and untreated cells (P < 0.05).
Figure 3
Figure 3
Frankincense essential oil-induced caspase apoptotic pathway in pancreatic cancer cells. An aliquot of 5 × 105 pancreatic cancer MIA PaCa-2 cells were seeded in each of 60 mm tissue culture plates. Adherent cells were then subjected to Fractions III (1:600 dilution) or IV (1:1,200 dilution) essential oil stimulation. Total cellular proteins were isolated between 0 (untreated control) and 4 h after treatment. Levels of pro- and activated-caspase-8, 9, 3 and PARP in MIA PaCa-2 cells were detected by Western blot analysis before and after frankincense essential oil treatment. Levels of β-actin was determined in the same samples and used as a protein loading control.
Figure 4
Figure 4
Frankincense essential oil-regulated signaling and cell cycle-related proteins expression in pancreatic cancer cells. Human pancreatic cancer cells (5 × 105) were plated into each of 60 mm tissue culture plates for adherence. Cells were treated with either Fractions III (1:600 dilution) or IV (1:1,200 dilution) essential oils. Total cellular proteins were isolated from these cells between 0 (untreated control) and 4 h following essential oil treatment. Levels of Akt and Erk1/2 proteins phosphorylation as well as total cyclin D1 and cdk4 proteins expression were determined using Western blot analysis, and compared between cells with and without frankincense essential oils treatment. Expression of β-actin was also assessed in parallel and used as a protein loading control.
Figure 5
Figure 5
Frankincense essential oil-suppressed pancreatic tumor growth in a heterotopic xenograft mouse model. The xenograft mouse tumor model was established by subcutaneous implantation of 1 × 107 MIA PaCa-2 cells in 250 μl HBSS supplemented with 20% Matrigel™ into the flank of each mouse. Tumors were allowed to grow for 7 days. Mice were randomly assigned to receive either 100 μl PBS (control; 5 mice) or Fraction IV frankincense essential oil (30 μl essential oil plus 70 μl PBS; 6 mice). Treatment was administered through subcutaneous injection away from the tumors. (A) Tumor volumes measured in situ. Arrows indicate the time of tumor size measurement followed by frankincense essential oil treatment. (B) Tumor volumes calculated after the tumors have been removed from the experimental subjects at the end of experiments. Images were taken at the time of tumor harvest. * indicates statistical difference between PBS- and essential oil-treated groups (P < 0.05).
Figure 6
Figure 6
Frankincense essential oil-induced anti-tumor activity in a xenograft mouse model. Harvested tumors were subjected to formalin fixation and paraffin embedding. Tumor blocks were cut at 5 μm thick, deparaffinized, rehydrated, and stained for cell growth and death markers. (A) Tumor cell growth. Tumor cells that are actively proliferating at the time of harvest were detected by PHH3 immunohistochemical staining. (B) Tumor cell death. Tumor cells undergoing apoptosis at the time of harvest were assessed by TUNEL staining. Images from 2 of each control and frankincense essential oil treated animals are presented (20x magnification); and inserts were taken at 60x magnification to highlight positive immunoreactivity. Immunoreactive cells were counted from a total of 20 randomly selected fields at 40x magnification from 2 separate sections from each animal. Results are presented as total numbers of PHH3- and TUNEL-positive tumor cells from these fields. * and ** indicate statistical differences between untreated and essential oil-treated groups at P < 0.05 and P < 0.01, respectively.

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