Screening Data Reveals that Spirogyra triplicata, a Fresh Water Algae Induces Robust Anti-Proliferative Activity Against A549 Cells

Cancer is a major health problem characterized by uncontrolled cell proliferation and its propagation.1 Statistical report of GLOBOCAN reveals that 18.1 million new cancer cases evolved within which 9.6 million cancer deaths eventuated in 2018 worldwide.2 Cancer treatment follows chemotherapy using cytotoxic drugs, radiation therapy and surgery.3 Though all these procedure have been reported to combat this dreaded disease, people still suffers with undesirable side effects.4 The use of plant-based bio-products for cancer treatment is rapidly growing in medical practices.5 Thus our vision was to hunt more natural sources for the treatment of cancer with minimum side effects. Scientists worldwide are engaged in designing the targeted therapies that can exterminate cancer cells without harming normal one. Natural products like algae,6 angeosperms,7-9 lichens,10 Mashroom 11 have been a continuous source of medicines to treat diseases and injury. Epidemiological studies proclaimed that the people of Asian countries who consumes high amount of fish and seafood have low incidence of particular type of cancers such as lung, breast, colorectal and prostate cancers.12 Reports consider algae as a repository of promising novel phytochemical as a source of biomedicine.13 Such therapies primarily try to understand how and by what mean cancer cells are different from normal non-transformed cells. It is explicit that cancer cells and normal cells are morphologically and behaviorally different. Current information suggests that bioactive components extracted from algae, as well as methyl jasmonate (a natural compound derived from the plant of jasmonates family), seem to have anti-cancer activities through multiple mechanisms of action, including downregulation of cancer-cell proliferation and metastasis, and through the promotion of apoptosis of cancerous cells.14 Many algae are still untouched to unmask. After getting positive results in antioxidant activity of some green algae, our focus was to check the antiproliferative activities of six green algae in human cancer and metabolomic studies of the most potent ABSTRACT Introduction: Algae comprise a promising source of novel components with potent therapeutic agents. In particular, algae have been considered as a potential source of new bioactive compounds. The antioxidant data of our previous study with six different algal methanolic extract reveals the presence of high antioxidant, total phenol content and total flavonoid content in Spirogyra triplicata. Thus, we further focused on screening the anti-proliferative activity of six different green algae on five different cancer cell lines like MCF7, A549, HEPG2, REH, MOLT4. Methods: To fulfill our aim we performed MTT assay for testing anti-proliferative activity and DAPI staining for observing nuclear morphology. We also looked into the metabolomic profiling of Spirogyra triplicata by GC-MS chemometric study. Results: The result indicates that after 24 hours of treatment with methanolic extract of Spirogyra triplicata A549 was the most sensitive cell line with IC50 value of 24.07 ± 1.09 μg/ml. Followed by Rhizoclonium fontinale and Hydrodictyon reticulatum with IC50 value of 25.97 ± 1.94 μg/ml and 32.50 ± 1.97 μg/ml respectively. The HEPG2 cell line was the second most sensitive cell line against S. triplicata with IC50 value of 30.20 ± 1.45 μg/ml. The MOLT4 cell line was detected as most resistant cell line against the green algal extract in this study. Though the methanolic extracts of six green algae showed maximum to moderate anti-proliferative activity on different cancer cell line but no significantly affect on normal PBMC was observed. Nuclear fragmentation was observed in a dose dependent fashion by DAPI staining on A549 cells treated with methanolic extract of Spirogyra triplicata. We further looked into the chemo profiling of Spirogyra triplicata by GCMS analysis. The result of GC-MS clearly indicates presence of nineteen major components and twenty-three minor components which have more or less bioactivity and would help in therapeutics in future. Conclusions: In brief this study indicates for the first time that green algae Spirogyra triplicata induces anti-proliferative activity specifically against A549 cell but not in normal PBMC. It can be concluded that Spirogyra triplicata holds a great promise as a good repository of anti cancer compounds which may be used in future drug discovery.


INTRODUCTION
Cancer is a major health problem characterized by uncontrolled cell proliferation and its propagation. 1 Statistical report of GLOBOCAN reveals that 18.1 million new cancer cases evolved within which 9.6 million cancer deaths eventuated in 2018 worldwide. 2 Cancer treatment follows chemotherapy using cytotoxic drugs, radiation therapy and surgery. 3 Though all these procedure have been reported to combat this dreaded disease, people still suffers with undesirable side effects. 4 The use of plant-based bio-products for cancer treatment is rapidly growing in medical practices. 5 Thus our vision was to hunt more natural sources for the treatment of cancer with minimum side effects. Scientists worldwide are engaged in designing the targeted therapies that can exterminate cancer cells without harming normal one. Natural products like algae, 6 angeosperms, 7-9 lichens, 10 Mashroom 11 have been a continuous source of medicines to treat diseases and injury. Epidemiological studies proclaimed that the people of Asian countries who consumes high amount of fish and seafood have low incidence of particular type of cancers such as lung, breast, colorectal and prostate cancers. 12 Reports consider algae as a repository of promising novel phytochemical as a source of biomedicine. 13 Such therapies primarily try to understand how and by what mean cancer cells are different from normal non-transformed cells. It is explicit that cancer cells and normal cells are morphologically and behaviorally different. Current information suggests that bioactive components extracted from algae, as well as methyl jasmonate (a natural compound derived from the plant of jasmonates family), seem to have anti-cancer activities through multiple mechanisms of action, including downregulation of cancer-cell proliferation and metastasis, and through the promotion of apoptosis of cancerous cells. 14 Many algae are still untouched to unmask. After getting positive results in antioxidant activity of some green algae, our focus was to check the antiproliferative activities of six green algae in human cancer and metabolomic studies of the most potent green methanolic algal extract. The result of which could be a great source of future medical discovery.

Isolation of peripheral blood mononuclear cells (PBMC)
Peripheral blood mononuclear cells constitute the cellular part of the blood containing all blood cells with a round nucleus. PBMCs are mainly invading cells that comprised of monocytes, T cells, B cells, natural killer (NK) cells, and dendritic cells. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood of healthy normal volunteers by percoll density (Sigma aldrich) gradient centrifugation (d=1.082 g/L) creating prominent layers containing specific cell populations. The cells can then be isolated by extracting the respective layer. The total cell number and the number of viable cells were determined with Trypan blue (Sigma aldrich) exclusion. PBMC were then recovered at the interface and the retrieved cells were washed and re-suspended in phosphate-buffered saline (PBS) and finally fixed in RPMI 1640 (Sigma aldrich) supplemented with 2mM glutamine(Sigma aldrich), gentamicin (Sigma aldrich) and 10% heat inactivated FBS (invitrogen). 15

Cell viability assay
To check the viability of cells after treating with methanol extracts, we performed the cell viability assay. Cells were harvested 96-well plate with increasing concentrations of STME and observed at 24 Hours of time point by MTT assay. 16 In brief cells were incubated in triplicata in a 96-well plate at different concentrations of methanolic algal extract in a final volume of 200µl for 24 hrs at 37°C. Three hours before the completion of time points, 20µl of MTT solution (5mg/mL) was added to each well. Formazone crystals formed, representing percentage of cell viability was assessed at 560 nm. Cell apoptosis (100%) was obtained by lysis of cells in 5% SDS lysis buffer. All treatments were performed in triplicata. The percentage of cell viability was calculated as mentioned below:

Nuclear staining with DAPI
Nuclear fragmentation is an important phenomenon of apoptosis and can be visualize by DAPI staining. 17 Cells were seeded in 12 well plate and treated with STME at different concentrations for 24h. After treatment, cells were collected, washed with 1 X phosphate buffered saline (PBS). DAPI (1μg/mL in PBS) was added and incubated for 15 min at room temperature. The stained cells were observed under a fluorescence microscope.

Microscopic staining of apoptotic cells by PI
Based on our MTT data, we found that A549 cell lines are mostly affected by Spirogyra triplicata. Thus we took control and three increasing concentration of STME treated A549 cell well plate. The treated 1×10 5 A549 cells were stained with freshly prepared PI (1μg/ml) and incubated for 10 min. after washing, the cells were visualized under fluorescence microscope. 18 (Dewinter).

Metabolomic study by GCMS Study
From our previous study, after getting positive antioxidant result and high amount of total phenol and flavonoid content, our next aim was to analyze the chemometric activity by GC-MS study.

Sample preparation for GC/MS analysis
The 10mg algal methanol extract was dissolved in 1 ml of the HPLC grade methanol, vortexed properly for well mixing and finally filtered through 0.22 μm syringe filter (Millipore Corp., Bedford, MA, USA).

Instrumentation and chromatographic conditions
Agilent Technologies 7890A GC System liquid autosampler was used for GC/MS analysis. The gas chromatograph was interfaced to a mass spectrometer instrument utilizing the following conditions viz. HP-5 ms column (30 m× 0.25 mm× 0.25 μm), fitted with silica and coupled to an Agilent triple quadrupole Mass selective Detector MSD7000, ionization voltage 70 eV; electron multiplier energy 2000 V; transfer line temperature,295°C. Helium as carrier gas (1 ml/min). The initial temperature was 60°C for 0 min, then gradually increased to 320 C automatically and in the split ratio mode (10:1), transfer line temperature 295 C. 19 Identification of components NIST MS search program v.2.2 (NIST 11) [National Institute Standard and Technology] was utilized for the interpretation GC/MS mass spectrum. Finally, name, molecular weight and structure of the components of the test material were determined.

Statistical analysis
The results were expressed as mean ± standard deviation. Descriptive statistics was used to analyze the mean, standard deviation, variation, and level of statistical significance between groups. When p < 0.05 and p < 0.01, it was considered statistically significant for analysis of percent inhibition of cell growth.

Differential anti-proliferative activity of algal extract by MTT assay
To fish out the most potent anti-cancerous extract, we investigated the anti-proliferative effects of six different green algal methanolic fraction and tested on five different cancer cell lines e.g. MCF-7, A549, HEPG2, REH, MOLT4. Each cell line was treated with six different algal extract for 24hour time points followed by MTT assay. Results of the MTT assay proved that A549 was the most sensetive cell line which was significantly down-regulated by most of the green algae ( Table 1). The most remarkable result was shown by Spirogyra triplicata methanol extract (STME) having maximum anti-proliferative activity against A549 cell line with IC 50 value of 24.07 ± 1.09 μg/ml. Followed by Rhizoclonium frontinale against A549 cell line, Spirogyra triplicata against HEPG2 cell line and Rhizoclonium frontinale against HEPG2 cell line with IC 50 value of 25.97 ± 1.94 μg/ml, 30.2 ± 1.45 μg/ml and 30.45 ± 2.67 μg/ml respectively. The anti-proliferative activity of A549 cell line mostly by Hydrodictyon reticulatum, Rhizoclonium crassipellitum and Pithophora cleveana methanolic extract with IC 50 values of 32.50 ± 1.35 μg/ml , 34.71± 1.79 μg/ml and 37.54 ± 1.47 μg/ml respectively. STME shows moderate anti-proliferative activity against MCF-7 cell line with IC 50 value 47.28 ± 1.98 μg/ml but against REH and Molt 4 cell lines did not show significant anti-proliferative activity having IC 50 values of 82.68 ± 2.38 and 89.71 ± 1.82 respectively (Figure 1b) . Rhizoclonium frontinale (RFME) also exhibited lower anti-proliferative activity against REH, and MOLT4 cells (Figure 1d). From the Figure 1 we can conclude that cancer cell viability decreased with the dose dependent fashion. However, anti-proliferative activity of Cladophora glomerata, Pithophora cleveana, Rhizoclonium crassipellitum, Hydrodictyon reticulatum were found to be of least anti-proliferative efficacies against MOLT4 cell line with IC 50 values of 72.82 ± 1.3 μg/ml, 202.8 ± 1.89 μg/ ml, 213.7 ± 2.83 μg/ml, 165.9 ± 1.34 μg/ml respectively (Table 1). Thus it is proved from the data that MOLT4 cells were most resistant (Table  1) to all the extracts tested. In a nutshell from the MTT assay results we observe that S. triplicata possesses most promising Anti-cancer potentiality followed by R. frontinale (Figure 1d) in A549 cells. Whereas no significant anti proliferative effect was observed in normal PBMC isolated from healthy donor.

Effects of S. triplicata methanolic extract on cellular morphological changes of A549 cells
As the methanolic extract of S. triplicata showed the most potential antiproliferative activity we further looked at the nuclear fragmentation of A549 cells upon treatment with S. triplicata. The characteristic nuclear fragmentation induced during apoptosis was measured using DAPI staining. DAPI is a nuclear stain that can be visualized under UV fluorescence when excited under fluorescence microscope. In our study, DAPI staining revealed the changes related with apoptosis in A549 cells treated with the methanolic fraction of S. triplicata. The morphological changes associated with apoptosis such as chromatin condensation, nuclear fragmentation, and margination of nucleus are evident in A549 cells upon treatment ( Figure 2).

Observation of A549 cells by PI staining
PI is a membrane impermeant stain; it can't penetrate through the living cell membrane. When the cell undergoes apoptosis, the cell membrane becomes leaky and the stain can enter into the cell. PI staining can be used to exhibit the morphologic condition of DNA after treatment with methanolic extract. Thus to observe the amount of dead cells we performed the PI staining. In our study four groups of cells were considered in which first one untreated group i.e. no STME concentration was given to A549 cells and three ascending concentration of STME were given to rest three cell groups. In control cells presence of little amount of PI stained cells were visualized which suggests that there was no such apoptotic cells. As most of the cells were alive, PI did not get into the cells. Whereas the number of PI stained cells increased according to the increase in the concentration of STME treatment (Figure 3a). The figure clearly represents the scenario where the control cell with little PI stained cell, means most of the cells having, membrane integrity, thus PI stain was unable to enter into maximum cells. Whereas the increasing concentration of STME changes the cellular membrane integrity thus allowed to enter PI within the cell and looks red in colour under fluorescence microscope and % cell death gradually increased (Figure 3b, 3c, 3d).

Chemo profiling analysis by GC-MS
Previous analysis with STME indicates higher percentages of antioxidant capacities as well as the total phenolic content and total flavonoid content. 13 Henceforth, it was further investigated for metabolomic profiling by GC/MS analysis. GC/MS chemometric profiling of STME as per aforesaid experimental procedure showed numerous different peaks indicating the presence of diversified phyto chemotypes ( Figure  4).  (Table  2a). Along with the nineteen major components, twenty-three minor   components were detected in trace amount (Table 2b, Figure 2). These nineteen major components were further investigated for their biological efficacies and most of them have diverse range of positive pharmaceutical and therapeutic functions [20][21][22][23][24][25][26][27] (Table 2). From the above study it can be concluded that the methanolic fraction of S. triplicata possesses a wide range of phytochemicals which holds a great promise for future drugs for combating cancer.

DISCUSSION
The use of naturally occurring plant-based products has shown promising results in the treatment of cancer. Due to the complexity of cancer, novel bioactive compounds with multi targeting efficacy are the need of the hour. In this paper we looked into the anti-proliferative effect of freshwater green algae Spirogyra triplicata by MTT assay. Previously we performed a pilot study with both Ethyl acetate and methanol extract of Spirogyra triplicata and our findings demonstrate that the methanolic fraction of S. triplicata possesses the most potent anticancer activity against human lung cancer cells (A549) and comparable activity in few other cancer cell lines. In contrast no significant cytotoxicity was observed in the normal PBMC cells. Next, we used DAPI, a fluorescent DNA-binding agent to visualize morphological changes of the nucleus. Nucleus gets fragmented with increase in concentration of the methanolic extracts. The GC/MS chemometric profiling is a useful technique to detect the volatile compounds present in natural products. The GC/MS fingerprinting of methanolic fraction of S. triplicata revealed the presence of nineteen major bioactive components in large amount along with twenty-three minor components in trace amount. The major phytochemical groups namely fatty acid esters, sterols,   unsaturated alcohols, alkynes etc. were found to be the dominant components and these are known for their positive biological functions. Neophytadiene , 2-Pyrrolidinone, 1-methyl-, Methyl tetradecanoate, Doconexent and Azulene were the major phytocomponents exhibiting bioactivity. The Neophytadiene shows antioxidant, analgesic antipyretic, anti-inflammatory, antimicrobial, antidiabetic, anti-inflamatoty, antiarthritic and anti-cancer activity. 2-Pyrrolidinone, 1-methyl-exhibits immunomodulatory activity and known as antimyeloma compound.
Methyl tetradecanoate is being used as a flavouring and fragrancing agent. Doconexent with mixture of other oil act as a very good supplement of DHA (which have anti inflammatory effect in brain). Azulene-acts as a volatile oil component. Data taken together reflects that methanolic extract of S. triplicata proves to be a good repository of phytochemicals that holds a great promise in the treatment of cancer in future. However, further in depth studies need to be performed to fully discover its anticancer properties by looking at the different cell signaling pathways linked with cancer development.

AUTHORS CONTRIBUTION
Ankita Mridha performed all assays and data collection. Priya K Gopal maintained the cell culture. Santanu Paul made over all data analysis and assessment of manuscript.