Abstract

The main objective of this study was to examine the anticancer activities of the gold nanoparticles of marine seaweeds viz., Gracilaria verrucosa, G. pudumadamensis and G. salicornia. Gold nanoparticles of the three species of Gracilaria were tested for their anticancer properties in vitro against HeLa and MCF-7 cancer cell lines. HeLa cell line is cervical cancer cells and MCF-7 is breast cancer cells. The anticancer activity of gold nanoparticles of the seaweed was observed based on cell viability and morphology of the treated cells and control. Among the three species, G. verrucosa showed a greater activity with an IC 49.80 against HeLa than the other two species. While greater anticancerous activity was observed in G. salicornia with IC 49.69 against MCF-7 cell line. The morphology of the treated cells showed a great variation when compared to the control cells. Thus, the in vitro assay indicated that the gold nanoparticles synthesized by these red seaweeds are the significant source of a noble anticancer agent. The study also revealed that G. verrucosa, G. pudumadamensis and G. salicornia could be the promising bioagents for cancer therapy in the near future.

1. Introduction

Seaweeds are a group of marine multicellular macroscopic algae mainly belonging to Chlorophyceae, Phaeophyceae and Rhodophyceae. They have gained much importance in food, pharmaceutical and agriculture industry. They are rich source of bioactive compounds which possess therapeutic properties. Seaweeds contain bioactive substances like proteins, lipids and polyphenols, with antibacterial, anticancer, antioxidant, antifungal, antiviral properties ^{1, 2}. Isolation of cytotoxic anti-tumor substances from marine organisms has been reported by several authors ^{3, 4, 5, 6, 7, 8}. Discovery and identification of bioactive compounds from natural resources is the most essential research in the present scenario. Hence in the present study, G. verrucosa, G. pudumadamensis and G. salicornia (Rhodophyceae) were collected from East coast of Tamil Nadu, South India to synthesize gold nanoparticles and to see its effect on anticancerous activity.

Algal nanotechnology has made its contribution to all spheres of human life. Globally biological green synthesize of nanoparticles using algae has attracted the focus of scientists from all fields mainly because the chemical methods are laborious process and are not eco-friendly. In the field of algal nanotechnology, synthesize of gold and silver nanoparticles is very popular and there are many research works carried out currently. However biosynthesize of silver nanoparticles and its effect as antibacterial, antiviral, anti-tumour is a more common work than biosynthesize of gold nanoparticles (AuNPs) ^{9, 10, 11, 12}. The biogenic synthesize of AuNPs using algae is a simple, low-cost, environmental friendly, nontoxic, reliable, and safe approach that can be used for a range of applications ¹³. The biosynthesized AuNPs have potential use as a carrier for anticancer drug delivery ¹⁴. Hence in the present work, AuNPs were synthesized from the red algae G. verrucosa, G. pudumadamensis and G. salicornia (Rhodophyceae).

Breast cancer is the most prevalent cancer in the world (22% of all the cases) and causes the highest percentage of the cancer deaths (14% of all cancer deaths) in women worldwide. HeLa is a cervical cancer cell line which has contributed to many medical breakthroughs. Though there are many advanced treatment for the cancer diseases, many studies have shown that some algae contain various biologically active substances with potential therapeutic applications in human ³. Algae are used as a valuable source of biomolecules for new drug development, including novel anticancer compounds ⁷. Hence in the present study, AuNPs synthesized from G. verrucosa, G. pudumadamensis and G. salicornia were used in the MTT assay to test their anticancer properties in vitro against the HeLa (cervical) and MCF-7 (breast) cancer cell lines.

2. Materials and Methods

Seaweeds were collected from the rocks of Tuticorin (GPS 8.7642° N, 78.1348° E) and from Rameswaram (GPS 9.2876° N, 79.3129° E). Collected samples were washed thoroughly with water to remove the debris and epiphytes. Based on the morphological and anatomical characteristics, they were identified with the manual published by Desikachary et al., (1998). The cleaned seaweeds were shade dried and powdered with mortar and pestle and sieved through a mesh size of <0.5 mm. Dried biomass powder was used for the synthesize of AuNPs. About 30 mg of seaweed powder was added to 10 ml of 10^-3 M aqueous HAuCl₄^- solution in a 20 ml test tube and incubated at room temperature. The colour change from yellow to deep red colour indicated the formation of AuNPs.

Synthesized AuNPs were extracted by centrifugation with 5,000 rpm for 15 min at 4°C. The crystalline AuNPs were used for MTT assay.

HeLa and MCF-7 cell lines were obtained from NCCS, Pune. The cells were maintained in Minimal Essential Medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a humidified atmosphere of 50 μg/ml CO₂ at 37°C.

Cells (1×10⁵/well) were plated in 24-well plates and incubated in 37°C with 5% CO₂ condition. After the cell reached the confluence, the various concentrations of the samples were added and incubated for 24 h. After incubation, the samples were removed from the well and washed with phosphate-buffered saline (pH 7.4) or DMEM without serum. 100 µl/well (5 mg/ml) of 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added and incubated for 4 h. After incubation, 1 ml of DMSO was added in all the wells. The absorbance at 570 nm was measured with UV-Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required for a 50% inhibition (IC 50) was determined graphically. The % cell viability was calculated using the following formula:

Graphs are plotted using the % of Cell Viability at Y-axis and concentration of the sample in X-axis. Cell control and sample control is included in each assay to compare the full cell viability assessments.

3. Results

The results showed that HeLa cell and MCF-7 cell proliferation was significantly inhibited by the AuNPs synthesized by G. verrucosa, G. pudumadamensis and G. salicornia. The results indicate that the sensitivity of HeLa cells for cytotoxic activity was higher against the AuNPs synthesized from G. verrucosa with IC value 49.80, G. pudumadamensis showed IC value 50.38. Comparatively the IC value was 52.62 for G. salicornia. The treated cells of MCF-7 cell line showed higher sensitivity to the AuNPs synthesized by G. salicornia with IC 49.69 whereas IC value of G. pudumadamensis was 50.15 and IC value was 51.72 for G. verrucosa (Table 1, Figure 1 - Figure 6). However IC 50 (concentration at which 50% of cells were dead) values were observed in almost all the three species of Gracilaria showing that all the three are potential anticancerous agents. In G. salicornia, treatment against MCF-7 cell line, the cell viability at 49.69% was observed at the concentration of 7.8 µg/ml and in G. verrucosa, treatment against HeLa cell line, cell viability at 49.80% was seen at the concentration of 31.2 µg/ml. However AuNPs of G. pudumadamensis also showed considerable activity at IC 50 against both HeLa and MCF-7 cell lines.

The plates were observed under an inverted microscope (Biolink) to detect morphological changes (Figure 7 - Figure 12). The results showed that HeLa cell and -7 cell proliferation was significantly inhibited by the AuNPs of G. verrucosa and G. salicornia. It was also observed that AuNPs of G. pudumadamensis also inhibited both HeLa and -7 cell proliferation. These results indicate that the sensitivity of human cervical cancer cell line and breast cancer cell line for cytotoxic drugs in the form of biosynthesized AuNPs from seaweeds, especially from Rhodophyceae members was high.

4. Discussion

There are reports on marine micro and macro algae prominently featured in cancer research. However most of these works are on the effect of different extracts of seaweeds using solvents such as ethanol, methanol and chloroform against cancer cell lines ^{15, 16}.

The present work is on the AuNPs synthesized from the marine algae Gracilaria sp. viz., G. verrucosa, G. pudumadamensis and G. salicornia. These AuNPs were treated against the HeLa cell line and MCF-7 cell line. In both the cell line treatments, all the three species of Gracilaria have shown 50% cell viability at varying concentrations. These AuNPs are not only cost effective but also simple and environment friendly. In the past few decades, several organo-gold complexes have emanated with hopeful antitumor, antimicrobial, antimalarial, and anti-HIV activities ¹⁷.

The need for the search of drugs against increasing cancer diseases which has no side effects has become inevitable. Synthesize of nanoparticles which are of biological origin are promising novel therapeutic agents for cancer treatments ¹⁸. Hence the present work on the effect of AuNPs on the two cancer cell lines prove that they can be used as therapeutic drugs for the cancer treatment.

5. Conclusion

Phytochemicals of marine algae such as hydroxyl, carboxyl, and amino functional groups, can serve as effective metal-reducing and capping agents to provide a robust coating on the AuNPs. Biosynthesize of nanoparticles using green resources is one of the promising researches in algae. This green method of synthesizing AuNPs plays a significant role as anticancerous therapeutic agent.

Abbreviations

AuNPs - Gold Nanoparticles, G- Gracilaria

References