Anti-Proliferative and Apoptotic Activity of Polyphenol-Rich Crude Methanol Extract of Gracillaria Edulis Against Human Rhabdomyosarcoma (Rd) and Breast Adenocarcinoma (Mcf-7) Cell Lines

Seaweeds are an important source of bioactive metabolites in the development of drugs and nutraceuticals. This study aimed to analyze the anti-proliferative and apoptotic activity of methanol extract (ME) of G.edulis on human Rhabdomyosarcoma (RD) and breast adenocarcinoma (MCF-7) cell lines. De-polysaccharide ME of G.edulis was extracted to determine the antiproliferative and apoptotic activity. The cytotoxic activity was evaluated by MTT and neutral red uptake assay, while apoptotic activity was characterized by cellular morphology, DNA fragmentation and Caspase 3/7 assay. The results of cytotoxicity assay showed that the decrease in the percentage of cell viability in a dose-dependent manner as signified by cell death. The apoptosis was induced by activating the Caspase 3/7 pathway and the RD cell line showed prominent activation of Caspase 3 compared to the MCF-7 cells have low activation. Further, the typical DNA ladder pattern was observed in RD cells, whereas MCF-7 cells did not show a typical ladder pattern. Thus, it can be concluded that the ME of G.edulis possesses anticancer activity via activating the Caspases pathway.


Introduction
Recently, the burden of different types of cancer increased significantly around the globe. The World Health Organization projects that, by 2035, the world could see 24 million new cancer cases and 14.5 million cancer-related deaths a year [1]. According to the Cancer Registry of Sri Lanka, about 23,530 cancer cases were reported in 2018, accompanied by 14013 deaths [2]). Breast cancer, skin cancer, and ovarian cancer are the leading cancers affecting women globally [1]. In addition, the soft tissue sarcoma is the third most common solid tumor in children [3]. Available cancer treatments cause several side effects with an increased risk of developing secondary cancers due to their long term potential toxic effects. Hence, it is vital to keep searching for an effective drug with minimum side effects that may benefit patients suffering from different types of cancer [4]. Among the natural resources, seaweeds play a major role in drug development due to the presence of bioactive secondary metabolites that exhibit potent biological activities such as antioxidant, anti-cancer, antidiabetic, etc. Gracillaria edulis (Gmelin) Silva is a red alga that belongs to the family Gracilariaceae. Polyphenols purified from red seaweeds are considered a rich source of antioxidants. In the search of new plant-derived drugs, natural antioxidant-rich marine seaweeds can be utilized to develop potential anti-cancer therapies. Therefore, the present study aimed to analyze the anti-proliferative and apoptotic activity of the methanol extract of G.edulis on human Rhabdomyosarcoma (RD) and human breast adenocarcinoma (MCF-7) cell lines.

Collection of Red Algae Gracillaria Edulis
The permission to collect the algae sample was obtained from the Department of Wild Life Conservation (permit number-WL/3/280/17). The red algae Gracillaria edulis was manually collected from Kalpitiya lagoon area, Puttalam (8°14'0" N, 79°46'0" E), Sri Lanka, and the specimen of algae sample was deposited at the Department of Zoology, University of Sri Jayewardenepura. The collected samples were cleaned and washed with fresh water to remove salt, sand, attached epiphytes, and organic matter. The samples were air-dried and ground into a fine powder and stored at −20°C until further use.

Preparation of The Methanol Extract
Homogenized Gracillaria edulis powder (10 g) was extracted three times using 70% methanol, subjected to sonication at 25 °C for three 90 min periods. The polyphenols were separated by precipitating crude polysaccharides using 70% ethanol (1:20 v/v). The supernatant was separated as polyphenol-rich methanol extract [5].

Maintenance of Cell Lines
The human rhabdomyosarcoma (RD) and breast adenocarcinoma (MCF-7) cell lines were obtained from the Department of Biochemistry, Faculty of Medical Sciences, University of Colombo, and cells were cultured in high glucose DMEM medium supplemented with 10% v/v FBS and 1% penicillin-streptomycin antibiotic solution. The cells were maintained in a 5% CO2 incubator at 37 °C.

MTT Assay
Cells were seeded in a 96-well plate at a density of 1 × 105 cells/well and incubated in FBS and antibiotic supplemented DMEM medium at 37 °C for 24 h. After incubation for 24 h, cells were treated with the different concentrations (10, 20, 30, 40, 50, 60 μg/mL) of the extract. Following 24 h of incubation, stock MTT solution (10 /~1 per 100/~1 medium) was added to all wells of an assay, and plates were incubated at 37 °C for 4 h. After incubation, 1 of 0.04 N HCI in isopropanol was added to all wells and mixed thoroughly to dissolve the dark blue crystals. The plates were read using SPECTRAmax-Gemini EM, Molecular Devices Inc, USA the Wavelength of 570 nm.

Neutral Red Assay
Cells were seeded in a 96-well plate at a density of 1 × 105 cells/well and incubated in FBS and antibiotic supplemented DMEM medium at 37 °C for 24 h. After incubation for 24 h, cells were treated with the different concentrations (10, 20, 30, 40, 50, 60 μg/mL) of the extract. Following 24 h of incubation, 0.04 mg/mL neutral red dye was added to all well and cells were incubated for 2-4 h. After the incubation, cells were washed with neutral red washing solution and rinsed with neutral red Solubilization Solution. The plates were read using SPECTRAmax-Gemini EM, Molecular Devices Inc, USA the Wavelength of 540nm.

In-vitro Cytotoxic Activity
The results of cytotoxic activity against the human RD and MCF-7 cells as determined by MTT and neutral red assays are shown in Table 1. The results exhibited a dose-dependent decrease in cell viability as signified by cell death. The extract of G.edulis exhibited potent cytotoxicity against RD (IC50: 49.86 ± 0.02 μg/mL) and MCF-7 (IC50: 34.43 ± 0.86 μg/mL) carcinoma cells when compared to the standard cycloheximide (IC50RD: 36.17 ± 1.78μg/mL; IC50MCF-728.76 ± 0.55μg/mL) as confirmed by the MTT assay. The neutral red assay, further, confirmed the potential cytotoxic activity of the extract (IC50RD: 33.47 ± 2.25μg/mL; IC50MCF-7: 35.13 ± 0.95μg/mL) against RD and MCF-7, compared to the standard cycloheximide (IC50RD: 32.78 ± 0.91μg/mL; IC50MCF-7: 27.84 ± 0.33μg/mL) which has ability to act as a protein synthesis inhibitor in eukaryotes.

Apoptotic activity
The apoptotic activity of RD and MCF-7 cells after 24 h of the treatment with the methanol extract was characterized by cellular morphology, DNA fragmentation, and Caspase 3/7 assay. Prominent activation of Caspase 3/7 was observed in the RD cells compared to the MCF-7 cells. Also, the highest Caspase 3/7 activity was observed after 4 h in RD cells, whereas in MCF-7 high Caspase activity was observed after 3 h of the treatment.
Similarly, the DNA ladder pattern was observed in RD cells treated with methanol extract and the standard cycloheximide whereas MCF-7 cells did not show any typical DNA ladder pattern ( Figure 2).
Further, apoptotic morphological features of RD and MCF-7 cells were observed using fluorescence staining (Hoechst stain) method in addition to the phase-contrast inverted microscope. As shown in Figure 3, treated cells with G.edulis methanol extract and standard staurosporine displayed typical morphological features compared to the untreated cells (control). RFU values of control: Control RD: 2933.9, Control MCF-7:3800.34). Net RFU value of the extract was calculated by subtracting the RFU values of the extract from the control.

Discussion
The present study showed the potent cytotoxic and apoptotic effect of methanol extract of G.edulis against human RD and MCF-7 cell lines. The G.edulis methanol extract can reduce the cell count (visible) and it caused the apoptotic response.
According to the cytotoxic assay, higher cytotoxicity was observed in the RD cells treated with the methanol extract of G.edulis compared to the MCF-7 cells as determined by MTT and neutral red assays. When comparing the cytotoxicity results, both MTT and neutral red assays showed more or less similar results as both assays measure the cell viability in different approaches. The MTT assay is based on the reduction of yellow tetrazolium MTT reagent to purple formazan crystals by cellular oxidoreductase enzymes present in mitochondria of viable cells whereas the neutral red assay is based on the accumulation of neutral red dye which is a eurhodin dye in the lysosomes of viable cells [8].
Lakshmi et al. (2016), found that the anti-proliferative activity of the methanol extract of G. edulis as 89.35% cell viability for 100 μg of methanol extract against MCF-7 cell line [9]. In the present study, 69.70% of cell viability was observed for 20 μg/mL of methanol extract. The observed high antiinhibitory activity of the present study might be due to the variation in extraction techniques as the present study extracted only the polyphenolic portion using the sonication method whereas the previous study was against the whole crude algae using the Soxhlet method for extraction.
Apoptosis is the programmed cell death that is regulated by different families of proteins. Among them, caspase is one of the important protein families involved in the cell death pathway (apoptosis). Caspase proteins are arranged in a cascade and coordinate the stereotypical events that occur during apoptosis by transmitting the death signal. Among the different types of caspases, caspase 3 is important in the process of apoptosis as it is activated by both intrinsic (mitochondrial) and extrinsic (death ligand) pathways. The activation of caspase 3 leads to the cleavage of different structural proteins such as poly(ADP-ribose) polymerase, caspase-activated DNase (ICAD), and RD-ME: 50 μg/mL RD-STANDARD: 30 μg/mL RD-CONTROL MCF-7-STANDARD: 30 μg/mL MCF-7-CONTROL MCF-7-ME: 30 μg/mL DNA fragmentation factor resulting in morphological alterations of cells such as cell membrane blebbing and cellular condensation. Further, DNA fragmentation apoptosis resulted in the cleavage of caspase-activated DNase (ICAD) [10].
In addition to caspase 3, caspase 7 is also an executioner protein of apoptosis, which is activated by cell death stimuli to induce apoptosis [11]. In the present study, a prominent elevation of Caspase 3/7 is observed in the RD cells after the 4 h of the treatment when compared to the MCF-7 cells. The results of the present study showed that the extract induces very low activation of Caspase 3/7 after 24 h of treatment. Further, Wang et al. (2016), confirmed that Caspase 3 is deficient in MCF-7 due to the partial deletion of the CASP-3 gene important for cell death, thus leaving the MCF-7 cells without typical apoptotic properties [12]. This was further confirmed by the DNA ladder pattern of the extract-treated MCF-7 cells. However, one band observed in the MCF-7 cells treated with the standard due to the activation of Caspase 7 causing cell membrane blebbing and DNA fragmentation [13].
During apoptosis, cells display typical morphological features such as cell membrane blebbing, the formation of the membrane-bounded vesicle, nuclear fragmentation, micronuclei formation, cellular shrinkage, cellular aggregation, the formation of cell clumps, and chromatin condensation. Most of these features were observed in both RD and MCF-7 cells treated with the methanol extract of G.edulis and standard cycloheximide compared to the untreated cells.

Conclusions
It can be concluded that the crude methanol extract of G. edulis exhibits significant antiproliferative and apoptotic activity against RD and MCF-7 cell lines. The RD cell line showed prominent activation of Caspase 3 compared to the MCF-7 cells have low activation.