Frankincense essential oil suppresses melanoma cancer through down regulation of Bcl-2/Bax cascade signaling and ameliorates heptotoxicity via phase I and II drug metabolizing enzymes

Abstract

Melanoma is a deadly form of malignancy and according to the World Health Organization 132,000 new cases of melanoma are diagnosed worldwide each year. Surgical resection and chemo/drug treatments opted for early and late stage of melanoma respectively, however detrimental post surgical and chemotherapy consequences are inevitable. Noticeably melanoma drug treatments are associated with liver injuries such as hepatitis and cholestasis which are very common. Alleviation of these clinical manifestations with better treatment options would enhance prognosis status and patients survival. Natural products which induce cytotoxicity with minimum side effects are of interest to achieve high therapeutic efficiency. In this study we investigated anti-melanoma and hepatoprotective activities of frankincense essential oil (FEO) in both in vitro and in vivo models. Pretreatment with FEO induce a significant (p < 0.05) dose-dependent reduction in the cell viability of mouse (B16-F10) and human melanoma (FM94) but not in the normal human epithelial melanocytes (HNEM). Immunoblot analysis showed that FEO induces down regulation of Bcl-2 and up regulation of BAX in B16-F10 cells whereas in FM94 cells FEO induced dose-dependent cleavage of caspase 3, caspase 9 and PARP. Furthermore, FEO (10 μg/ml) treatment down regulated MCL1 in a time-dependent manner in FM94 cells. In vivo toxicity analysis reveals that weekly single dose of FEO (1200 mg/kg body weight) did not elicit detrimental effect on body weight during four weeks of experimental period. Histology of tissue sections also indicated that there were no observable histopathologic differences in the brain, heart, liver, and kidney compare to control groups. FEO (300 and 600 mg/kg body weight) treatments significantly reduced the tumor burden in C57BL/6 mice melanoma model. Acetaminophen (750 mg/kg body weight) was used to induce hepatic injury in Swiss albino mice. Pre treatment with FEO (250 and 500 mg/kg body weight) for seven days retained hematology (complete blood count), biochemical parameters (AST, ALT, ALK, total bilirubin, total protein, glucose, albumin/globulin ratio, cholesterol and triglyceride), and the level of phase I and II drug metabolizing enzymes (cytochrome P450, cytochromeb5, glutathione-S-transferase) which were obstructed by the administration of acetaminophen. Further liver histology showed that FEO treatments reversed the damages (central vein dilation, hemorrhage, and nuclei condensation) caused by acetaminophen. In conclusion, FEO elicited marked anti-melanoma in both in vitro and in vivo with a significant heptoprotection.

Keywords: apoptosis; essential oil; frankincense; melanoma; tumor remission.

Figure 1. Cytotoxicity of FEO on B16-F10, FM94 and HNEM cells.

(A) B16-F10 cells; (B) FM94 cells; (C) HNEM cells; (D) FM94 cells treated with FEO (A-Untreated cells; B-3 μg/ml; C-5 μg/ml; D-7 μg/ml; E-10 μg/ml; F- Dox 5 ug/ml) for 24 h and morphological image was photographed by EVOS image analyser; E: HNEM cells treated with FEO (A-Untreated cells; B-3 μg/ml; C-5 μg/ml; D-7 μg/ml; E-10 μg/ml; F- Dox 5 ug/ml) for 24 h and morphological image was photographed by EVOS image analyser. Data presented as mean ± SD of triplicates of three independent experiments. ^*Represents significant difference at p < 0.05 compared with control. ^**Represents significant difference at p < 0.01 compared with control. ^***Represents significant difference at p < 0.001 compared with control. NS: Non significant. Scale bar indicates 10 um.

Figure 2. FEO induced nuclear fragmentation in B16-F10 cells: B16-F10 cells were treated with different concentrations of FEO for 24 hours and stained with hoechest stain 33258.

(A) Control; (B) 5 ug/ml; (C) 7 ug/ml; (D) 10 ug/ml. The nuclei were observed using fluorescence microscope (20 um).

Figure 3. FEO induce DNA fragmentation in B16-F10 cells.

B16-F10 cells were treated with 0, 5, 7 and 10 μg/ml for 24 hours. DNA was isolated resolved in agarose gel and examined by ethidium bromide staining.

Figure 4. Flow cytometric analysis of apoptosis: B16-F10 cells treated with FEO at 10 μg/ml for 24 h.

Annexin-V FITC and propidium iodide staining were used to analyze apoptosis by flow cytometry. (A) Untreated cells showed 2–3% of cells in early or late apoptosis stage. (B) FEO treated cells, significant increase (^*P < 0.05) in percentage distribution of cells in early (25%) and late (10%) apoptosis were observed.

Figure 5. Molecular mechanism of FEO induced apoptosis in B16-F10 and FM94 cells.

(A) Bcl-2 and Bax protein expression of B16-F10 cells was determined after 24 and 36 h of treatment with 10 μg/ml of FEO; (B) Cleaved Caspase 9, Caspase 3 and PARP expression of FM94 cells was determined after 24 h treatment with 0, 7, 10 μg/ml of FEO. PC indicates positive control (5 μg/ml of doxorubicin); (C) MCL-1 expression of FM94 cells was determined after 3 h, 6 h, 12 h, and 24 h treatment with 10 μg/ml of FEO. Actin used for normalization of protein expression.

Figure 6. Effect of FEO on animal body weight: Data presented as mean ± SD (n = 8).

Mice were treated with FEO (1200 mg/kg body weight) and observed for 30 days at weekly intervals.

Figure 7. In vivo of toxicity of FEO on major organs: Mice were treated with FEO (1200 mg/kg body weight).

At the end of experimental period major organs such as brain, heart, liver and kidney were excised and stained with hemotoxylin and eosin. Scale bar indicates 10 μm.

Figure 8. Melanoma tumor remission efficacy of FEO: Data presented as mean ± SD (n = 6).

Tumor was induced by transplanting B16F10 cells (5 × 10⁵) in C57BL/6 mice. Tumor bearing mice treated with FEO (300 and 600 mg/kg of body weight) every two days for the total experimental period of 14 days and volume of tumor was measured. FEO 300 mg/kg bwt significantly (^*p < 0.05) reduce the tumor volume on day 14 compare to control group. FEO 600 mg/kg bwt significantly (^**p < 0.01, ^***p < 0.001) reduced tumor burden on day 10, 12, and 14 compared to control group.

Figure 9. Efficacy of FEO on acetaminophen induced hepatic injury: Mice were divided into five groups (n = 6).

(A) Untreated control: Mice received normal saline (0.9% v/w) i.p. for seven days. (B) Acetaminophen treated Acetaminophen (750 mg/kg body weight) alone injected to mice i.p. for seven days to induce hepatic injury. (C) Frankincense 250 mg: Mice treated simultaneously with Acetaminophen (750 mg/kg body weight) and FEO (250 mg/kg body weight) for seven days. (D) Frankincense 500 mg: Mice treated simultaneously with Acetaminophen (750 mg/kg body weight) and FEO (500 mg/kg body weight) for seven days. (E) Silymarin 25 mg: Mice treated simultaneously with Acetaminophen (750 mg/kg body weight) and silymarin (25 mg/kg body weight) (positive drug control). After seven days of treatment period the liver of all the animals was excised and histology was carried out. Scale bar indicates 10 μm.

Figure 10. Efficacy of FEO on biochemical parameters of acetaminophen induced hepatic injured mice: Values are expressed as Mean ± SD (n = 6).

^*p < 0.05 is considered significant when compared with group I; ^**p < 0.01 is considered significant when compared with group II by Dennett’s multiple comparison test. Group I: Untreated control fed orally with normal saline (0.9% v/w) 5ml/kg body weight daily. Group II as hepatotoxic control treated with acetaminophen (750 mg/kg body weight). Group III and IV received FEO 250 and 500 mg/kg body weight respectively along with acetaminophen (750 mg/kg body weight) for seven days. Group V treated with Silymarin (25 mg/kg body weight) with acetaminophen (750 mg/kg body weight) for seven days. (A) AST, ALT, ALK. (B) Total bilirubin and total protein. (C) Cholesterol, glucose, and triglyceride. (D) Albumin/globulin ratio.

Figure 11. Efficacy of FEO on drug metabolizing enzymes of acetaminophen induced hepatic injured mice: Values are expressed as mean ± SD (n = 8) animals.

^*p < 0.05, ^**p < 0.01 represent significant changes against control. Group I: Untreated control and fed orally with distilled water daily for seven days. Group II: Mice treated with FEO 250 mg/kg body weight. Group III: Mice treated with FEO 500 mg/kg body. Group IV: Mice treated with 0.75% BHA along with diet (positive control). (A) CytP450 and Cytb5, (B) GST. BHA – butylated hydroxyanisole; Cyt P450 – Cytochrome P450; Cyt b5 – Cytochrome b5; GST – Glutathione S-transferase.