Evaluation of anticancer activity of Melaleuka alternifolia (i. e. tea tree oil) on laryngeal cancer cell line (Hep2) - an in-vitro study

Introduction

Laryngeal cancer is the ninth most common cause of cancer in males in Asia and seventh most common cause of cancer in males in India respectively. In 2012, an estimated 25,446 new cases were diagnosed, and 17,560 Indians lost their lives from laryngeal cancer1. In India, the incidence of laryngeal cancer has been reported to be 1.268.18 per 100,000 population, in different regions in the country.² This contributes to approximately 3-6% of all cancers in men. The 5-year survival for laryngeal cancer in India is approximately 28%.  Epidemiological studies carried out on laryngeal cancer have highlighted the peculiarities of the disease, like varying risk factors and wide regional variation in incidence and survival percentage. Indian studies show tobacco, alcohol, long term exposure to indoor air pollution, spicy food, and non vegetarian diet as risk factors for laryngeal cancer.³

As far as the treatment it indicate that a treatment strategy is concerned involving induction chemotherapy and definitive radiation therapy can be effective in preserving the larynx in a high percentage of patients, without compromising overall survival.

Currently development and search for novel and effective anti cancer agents have become very important issues.⁴ To date, many natural products isolated from plant sources have been investigated for the discovery of the potential anticancer effects. Higher plants have long been shown to be excellent and reliable sources for the development of novel anti cancer drugs. One such naturally available plant extract tea tree oil (Melaleuka alternifolia), especially its anticancer activity, has not yet been investigated in Laryngeal cancer. Hence, this in vitro study aims at evaluating anticancer activity of this plant extract on Laryngeal cancer (Hep2) cell line. For control we used Monkey larynx (Vero) cell line. Also we decided minimum IC50 value of TTO on Laryngeal cancer (Hep2) cell line.

Material and Methods

Source of data

Before the start of the study ethical clearance was obtained by Institutional Review Board. We evaluated the anticancer activity of M.alternifolia (TTO) on Laryngeal cancer (Hep2) cell line and Monkey Kidney (Vero) cell line which was used as control in our study. Both The cell lines were procured from NCCS  National Centre For Cell Science, Pune.  Then it was subjected for MTT Assay to assess the cell viability and cell cytotoxicity (Cell Lysis).

MTT Assay

1. MTT solution preparation (stock solution): 5 mg in 1 ml of PBS.

   MTT (yellow dye) reduced by succinic dehydrogenase in the mitochondria of viable cells to purple formazan crystals.Formazan crystal production is directly proportional to the viable cells and inversely to the degree of cytotoxicity. When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells.

2. Cell culture :

   The cell line used for the study were Hep2(human procured from NCCS, Pune) and Vero cell line(Normal healthy Monkey Kidney cell line used as control procured from NCCS, Pune).The cell lines were maintained in 96 wells micro titer plate containing MEM media supplemented with 10% heat inactivated fetal calf serum (FCS), containing 5% of mixture of Gentamicin (10ug), Penicillin ( 100 Units/ ml) and Streptomycin (100µg/ml) in presence of 5% CO2 at 37ºC for 48-72 hours.

Cytotoxicity Assay:

In vitro growth inhibition effect of test compound was assessed by calorimetric or spectrophotometric determination of conversion of MTT into “ Formazan blue” by living cells. Remove the supernatant from the plate and add fresh MEM solution and treat with different concentrations of extract or compound appropriately diluted with DMSO. Control group contains only DMSO. In the current study, 10, 20, 25, 30 and 50ul of the stock solution (10mg / ml prepared in DMSO) were added to respective wells containing 100ul of the medium. So, the final concentrations were 10, 20, 25, 30 and 50ug / ml. That means to say the various concentration of TTO used to evaluate its anticancer activity were 100%, 50%, 25%, 12.5%,6.25%,3.125%, 1.562%, 0.781%, 0.390%, 0.195% respectively.

A)  After 24hrs incubation at 37ºC in a humidified atmosphere of 5% Co2, stock solution of MTT was added to each well (20µl, 5mg per ml in sterile PBS) for further 4 hr incubation.

B)  The supernatant carefully aspirated, the precipitated crystals of “Formazan blue were solubilised by adding DMSO (100µl) and optical density was measured at wavelength of 570nm by using LISA plus.

3. The results represent the mean of five readings.

The concentration at which the OD of treated cells was reduced by 50% with respect to the untreated control.

Formula:

Surviving cells (%) = Mean OD of test compound ×100

                                           Mean OD at control

Principle of assay

This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product. The cells are then solubilised with an organic solvent (eg. DMSO, Isopropanol) and the released, solubilised formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells5. 

Results

The Fig 1 and 2 depict the various concentration of TTO when treated with Hep2 cell line showing Min IC50 value of 0.781µg/ml.

The Fig 3 and 4 depict the various concentration of TTO when treated with Vero cell line showing viability of cells more than the Hep2 cell line. The cell inhibition was noted to be 35.19 with 50% concentration levels.

Table 1 shows Spearmans Correlation Coefficient Correlation with P value <0.001 indicating significant results when TTO was treated with Hep2 and Vero cell line.

Discussion 

TTO was used since 1920 by Australian aboriginal soldiers for treating wound infections in their kit as one of the element during the world war II. Whereas crushed leaves were used for treating cold, cough and as other skin ailment. Penfold  in 1920 invented more than 100 components of TTO The major are: terpinen-4-ol, γ-terpinene, α-terpinene and 1,8-cineole.⁶

We received this commercially available Tea tree oil from Crystal aromatics New Delhi, imported from Australia with Refractive index 1.475, at 250 weight/ml was 0.8850gm/ ml. This oil is obtained through the steam distillation process and is always stored in amber colored bottle to prevent oxidation.

The results mention that when the TTO was treated with Hep2 cell line There was 50% of cell lysis noted with Min IC50 O.781µg/ml .Whereas the maximum cell destruction with Normal control i.e Vero Cell line noted to be 35.19% at the concentration level of 50% of TTO. This shows that the cell destruction was noted to be quite higher when TTO was treated with Hep 2 cell lie than the Vero cell line.

Taxol⁷ from the bark of the pacific yew tree, E. tirucalli latex8, Nigella sativa⁹, (or black cumin), Polyacetylene capillin10 are the other naturally available plant extracts which are considered to quite effective anticancer agents on Hep2 Cell line. But the efficacy of TTO as an anticancer agent with its MIC50 in our study has shown better results than the other naturally available plant extracts from the previous literature.

This variation in the result could be due to differences in the method and concentrations used from the previous studies.

Conclusion

Currently, a great deal of research is focusing on the investigation of certain plant species as a source of experimental therapeutic agents, specifically with medicinal values in treating cancer. Hence, this Min IC50 value of TTO with its greater efficacy related to its anticancer activity can be brought to the level of clinical trials in the coming future.