INTRODUCTION:

Cancer is one of the life threatening health problems across the world. Based on statistics, 8.8 million deaths were reported worldwide in 2015 due to cancer. It is estimated that approximately 26 million new cases will be reported by 2030, and the quantum would increase in the future.¹ Tumour results due to the uncontrolled proliferation of cells which resist the cells from undergoing apoptosis along with spreading the abnormal cells leading to the death of the individual. The available chemotherapeutic drugs kill not only the cancer cells but also affect the normal cells causing serious side effects.² Hence drug discovery from natural sources with lesser side effects and highly specific is a major scientific pursuit. Several anticancer drugs have been isolated from plants, microorganisms and the marine organisms.³

Marine algae serve as inexhaustible resources of bioactive secondary metabolites with activities ranging from anti diabetic, anti oxidant, anti inflammatory and anti tumour activity.^4,5 Chances for finding novel molecules of great therapeutic potential, specifically anti cancer activities are quite high among the marine algae. Hence, in the present study, we focussed to explore the marine alga, Turbinaria ornata, for anti cancer applications in our pursuit of finding an efficient anticancer lead molecule. T.ornata is seen in the intertidal region of the sea and normally attached to the substratum with the hold fast. It is rich in secondary metabolites.⁶ Extensive biological activity studies from Turbinaria species have been reported by researchers. But till today no compound has been approved by FDA for anti cancer applications from this group.

MATERIALS AND METHOD:

Sample collection and preparation of extracts:

The seaweed, T.ornata was collected from the Mandapam camp, Ramanathapuram district, Tamil Nadu in India. The collected specimen was stored in cold storage and a small portion of the specimen was stored in 4% formalin for identification. The seaweed was identified as Turbinaria ornata by Dr. Eswaran, Principal scientist in Marine Algal Research station at Madapam camp (Figure. 1). Using methanol, it was extracted in a soxhlet apparatus which was concentrated using a rotary evaporator at 40ºC. The crude extract was stored at 4ºC until use and a stock solution of 1 mg/ml in DMSO was used for the assays.

Figure 1. Macroscopic view of Turbinaria ornata

Purification of the methanol extract using thin layer chromatography:

The methanol extract of T.ornata was partially purified by commercially available silica coated TLC sheets (50×20 cm size, Merck).

Anti proliferative assay:

MTT assay:

Cell lines used in our experiment were Cervical cancer cell line (HeLa cells), Liver cancer cell line (HepG2), and breast cancer cell line (MCF-7) procured from NCCS, Pune. These cells were subcultured and seeded onto 96 well plates and incubated for 24hrs for cell adherence. Various concentrations of the samples (1.0, 10.0, 50.0 and 100.0μg/ml) were added to the cells (1 x 10⁶ cells/ml) for 24 hr, 48hr and 72 hrs. After the treatment period, MTT assay was performed as per the standard protocol.⁷

Trypan blue exclusion assay:

The cell concentration and viability of the bioactive fraction treated cancer cells were determined by trypan blue method.⁸ Cells (1 x 10⁶ cells/ml) were sub cultured in 25cm² culture flasks, IC₅₀ concentration of the sample was added to the cells and kept for 48 hrs of incubation. The cells were trypsinized and collected by centrifugation. 5μl of cell suspension with equal volume of trypan blue dye were taken and the cells were counted in a haemocytometer under 40X of a binocular microscope (Labomed, Germany).

LDH assay:

The membrane integrity of the treated cells was analysed on the basis of release of cytosolic enzyme LDH (lactate dehydrogenase assay). LDH activity of the cells was performed as per the instructions provided in the LDH assay kit.⁹

Caspase assay:

Caspase activity was analysed using the Caspase Colorimetric Assay Kit (G Biosciences, kit 786-205A) as per the instructions given in the manual. Cells were treated with IC₅₀ concentration of the sample for 48 hrs and caspase activity was checked after this duration.

Cell cycle analysis:

The cell cycle stages of the cancer cells (1 x 10⁶ cells/ml) treated with TF7 fraction were assessed by flow cytometry method with the help of propidium iodide staining.¹⁰ The cells were processed after 48 hrs of treatment, and the pellet was washed twice with PBS and the cell pellet was resuspended in ice cold buffer (in tubes coated with 2% BSA in PBS overnight). The cell suspension was transferred into 9ml of ice cold 70% ethanol and stored in - 40ºCfor later use. The cell pellet was stained with propidium iodide (PI final concentration - 40μg/ml) and incubated for 15 minutes under the dark. The distribution of the DNA content in the treated cancer cells was analysed in BD FACSVerse.

DNA fragmentation:

Genomic DNA was isolated from the treated cells. The assay was performed based on the instructions given in the kit manual (Bangalore, Genei).

GC MS analysis:

The bioactive fraction TF7 was subjected to GC MS analysis to separate the compounds present in it. The fraction was injected into the instrument at (Central Silk Technology Research Institute, Bangalore) in which the fraction was separated based on the mass- to -charge ratio. The components produced different peaks at different retention times. The unknown compounds in the sample were quantified with the help of library search.

RESULTS:

The methanol extract of T. ornata was evaluated for anticancer activity against various cancer cell lines (cervical cancer cells, breast cancer cells and liver cancer cells). In our results, we found that the methanol extract of sample demonstrated good cytotoxic effects at 100μg/ml concentration against all the cancer cells (Table 1). The highest effect was seen in HeLa cells treated with the extract at 100μg /ml concentration after 72 hrs with a viability of 50.2%, followed by HepG2 cells with a viability of 55.8%.

Table 1 : Percentage viability of human cancer cell lines treated with T. ornata extracts by MTT assay

Concentration (μg/ml)	HeLa				HepG2				MCF-7
	Control	Treated			Control	Treated			Control	Treated
	Control	24h	48h	72h	Control	24h	48h	72h	Control	24h	48 h	72 h
1	100	94.31	93.88	71.65	100	100	100	89.56	100	91.73	97.85	85.29
10	100	95.17	93.18	65.69	100	87.80	82.99	79.96	100	87.73	84.82	77.87
50	100	71.20	79.69	63.26	100	71.71	69.87	59.25	100	84.53	83.57	69.56
100	100	66.03	63.69	50.24	100	68.76	62.70	55.89	100	83.86	70.53	66.75

Figure. 2 Effect of TF7 fraction as per MTT assay on a) HeLa cells b) MCF-7 cells and c) HepG2 Cells post treatment with fraction TF7 for 24, 48 and 72 hrs. *represents significance at p<0.05, **represents significance at p<0.01.

The methanol extract of T.ornata was further fractionated by thin layer chromatography using the solvent system hexane: ethylacetate: chloroform (0.9:0.6:1.2). Nine different bands got separated which were visible under the UV and visible lights. These fractions were screened for cytotoxicity by MTT assay in order to select the fraction having highest activity. Fraction 7 (TF7) showed maximum cytotoxic effects against the cancer cells with a viability of 40.48% in HeLa cells, 42.12% in MCF-7 cells and 45.78% in HepG2 cells at 50μg/ml concentration after 72 hrs (Figure 2). The IC₅₀ concentration of TF7 fraction was 40μg/ml in the case of MCF-7 cells followed by HeLa cells with 41μg/ml and HepG2 cells with 46μg/ml at 72 hrs of treatment.

As per trypan blue assay ( Table 2), the cell count of the TF7 treated MCF-7 cells was found to be drastically reduced from 12.7 x 10⁶ cells/ml in the controls to 4.06 x 10⁶ cells/ml (with 32.4% viability), that of HepG2 cells reduced from 10.3 x 10⁶ cells/ml in controls to 8.8 x 10⁶cells, (57.7% viability) and that of HeLa cells reduced from 3.7 x 10⁶cells/ml to 3.1 x 10⁶ cells/ml (54.4% viability), thus demonstrating anti proliferative property.

Table 2 Total cell count and percentage viability as per trypan blue analysis of cells treated with fraction TF7

Cell lines	Control		Treated
Cell lines	Cell count (1x 10⁶cells/ ml)	Viability (%)	Cell count (1x10⁶ cells/ ml)	Viability (%)
MCF-7	12.69	99.29	4.06	32.42
HepG2	10.30	98.47	8.82	57.72
HeLa	3.72	97.38	3.16	54.48

The mechanism underlying the anti proliferation was analysed by other in-vitro assays. Cell damage in the treated cells was analysed by measuring the release of LDH enzyme to the media. As per the results of this assay, the cytotoxicity was found to be 62.37% in HeLa, 54.92% in MCF-7 and 43.58% in HepG₂cells (Figure 3A). Apoptosis mediated by the fraction in the cancer cells was determined by caspase and DNA fragmentation assays. Treatment with the IC₅₀ concentration of the fraction caused an increase in the level of caspase enzyme activity with 46.21% in HeLa, 10.32% in MCF-7 and 25.41% in HepG2 cancer cells as compared to that of untreated control cells (Table 3).The ongoing apoptosis was validated in the treated cancer cells from the DNA fragmentation results. The DNA from the treated cells showed a smearing pattern on agarose gel electrophoresis whereas the DNA from control cells were intact and formed a single band (Figure 3B). DNA fragmentation is considered as the biochemical hallmark of apoptosis.

Table 3 : Caspase activity of Hela, MCF-7 and HepG2 cells treated with TF7 fraction from T.ornata

Samples	HeLa cells
Samples	Initial activity (%)	Final activity (%)	Increase in caspase activity (%)
Control	3.78	15.15	11.22
Treated	11.36	57.57	46.21
	MCF-7 cells
Control	14.19	20.64	6.45
Treated	25.80	36.12	10.32
	Hep G2 cells
Control	8.28	33.14	24.86
Treated	9.94	35.35	25.41

When the cancer cells treated with the fraction TF7 were injected to flow cytometry, it was found that there was cell cycle arrest at G1 phase with higher proportion of HeLa and MCF-7 cells in the G1 phase with 87.68% in HeLa and 77.43% in MCF-7 (Figure 4). Lesser number of cells were observed in G2 phase ( 1.49% in HeLa and 6.32% in MCF-7 cells) when compared to control group. In case of HepG2 cells, higher proportion of apoptotic cells (10.8%) were accumulated in G2 phase after TF7 treatment. G2 phase cells increased from 7.86% to 10.89% along with decrease of S phase from 19.62% to 17.43%. This indicates G2 phase arrest in HepG2 cells and the cell line specificity of TF7 fraction in inducing apoptosis.

Characterization of this promising TF7 fraction through GC MS analysis, resulted in indicating the presence of 2- phenoxy ethanol compound in the fraction TF7, which is reported to have anti microbial effects.¹¹ The presence of this bioactive compound might be responsible for the anti cancer activity of T.ornata (Figure. 5A & B).

Figure. 5(A) GC MS analysis of fraction TF7 (A).The chromatogram of the fraction TF7 showing peaks at different retention times (RT) (B). Mass spectrum of the fraction at RT 8.766 along with structure of 2-Phenoxy ethanol.

DISCUSSION:

Natural compounds from marine source such as the seaweeds show immense promise towards cancer therapy due to the structurally diverse bioactive secondary metabolites present in them.¹² Seaweeds are well known for their biological activities such as anti-oxidant, anti cancer, anti-inflammatory, anti-microbial and anti viral activities.¹³ In many research articles the anti cancer activity of the crude extracts of various brown algae against cancer cell lines were documented.¹⁴ Owing to the lack of specificity and efficacy of the current treatments for cancer, in the present study, we focussed to analyse the anti cancer potential of the marine alga T.ornata. There are many in vitro studies documenting the antiproliferative activity of brown seaweeds against different human cancer cells.^15,16,17,18 Through the current study we found that T.ornata has good cytotoxic effects to the cancer cells at lower concentrations (50µg/ml), while in an earlier study, it was reported that the hexane extract and acetone extracts (TO-HE and TO- AE) having their efficacy concentrations as 62.91, 93.00 and 72.6 and 106.6 mg/ml.¹⁹

In normal healthy tissues apoptosis or programmed cell death prevents the propagation of faulty DNA unlike the cancer cells which evade apoptosis. Hence, in cancer drug discovery studies, compounds which induce apoptosis are crucial. In our present study, the bioactive fraction TF7 from T.ornata was triggering apoptosis in the treated cancer cell as evidenced by DNA fragmentation and elevated caspase activities. It was reported earlier²⁰that fucoidan from the brown algae inhibits growth and induces apoptosis in human colon cancer cells, which was mediated by the activation of caspases. Cell cycle mechanism is one of the main mechanisms to find out the differences between normal and cancer cells. In normal cells, the cell cycle is controlled by a complex series of signaling pathways by which a cell grows, replicates its DNA and divides. This process also includes mechanisms to ensure corrections in faulty DNA and if not, the cells commit suicide. But in cancer cells, this cell cycle regulation doesn't exist and the cells grow unconditionally along with the proliferation of the mutated gene. In our study, we found that TF7 treatment to cancer cells resulted in the arrest of cell cycle at both G1 and G2 phase, thus leading to their death. This indicates that fraction TF7 has very good anti proliferative effects to the cancer cells.

In earlier reports of marine brown algae on cancer cells, anti proliferative activity was checked only in the crude extracts. In our present study, we partially purified the compound through TLC fractionation. Based on our results, we report the anti cancer activity of the selected fraction TF7 against three different cancer cell lines (HeLa, MCF-7 and HepG2 cells). Further characterization through GC-MS revealed the presence of 2- Phenoxy ethanol compound in the Turbinaria ornata which was documented as an anti microbial compound. 2- Phenoxy ethanol is a glycol ether used as a perfume fixative and antiseptic solvent. Phenoxyethanol is an ether alcohol with aromatic properties. Phenoxyethanol acts as an effective preservative in pharmaceuticals, cosmetics and lubricants.²¹ It is reported to have anti microbial activity and also used as a vaccine to inactivate the bacteria.¹¹ The bioactive compounds in T. ornata include essential fatty acids, essential oils, flavouring agents, plasticizer, fungicides and insecticides. These compounds are beneficial to mankind in various aspects.²² Through the results of our present study on T. ornata, we could confirm the anti cancer bioactivity of TF7 fraction and further in-vivo studies are warranted for drug development.

CONCLUSION:

It can be concluded that the marine brown alga Turbinaria ornata has potent anti proliferative activity against human cancer cell lines which show promise towards future studies focussing on pre clinical, clinical and drug developmental aspects.

ACKNOWLEDGEMENT:

The authors are thankful to Jain University for providing the fellowship and for the infrastructural facilities to carry out the work. The authors are grateful to Dr. K. Eswaran, Principal Scientist in Marine Algal Research station for identifying the seaweed used for the work.

The authors are grateful to Dr. M.A. Joseph, Scientist at Central Silk Technological Research Institute, Bengaluru, for providing GC-MS facility and analysis of the samples in his institute.

CONFLICT OF INTEREST:

The authors declare that there are no conflict of interest.

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Received on 20.11.2019 Modified on 10.01.2020

Research J. Pharm. and Tech 2020; 13(9):4263-4268.

DOI: 10.5958/0974-360X.2020.00752.0


Figure 3(A). LDH cytotoxicity of fraction TF7 treated cancer cells. (B). DNA fragmentation analysis of sample treated with TF7