Anticancer potential and cytotoxic effect of some freshwater cyanobacteria

Purpose: To investigate the anticancer potential and cytotoxicity of some freshwater cyanobacterial extracts on Human Colon Carcinoma (HT29), Rat Brain Glioma (C6), Human Cervix Carcinoma (HeLa), Human Lung Carcinoma (A549), Human Breast Adenocarcinoma (MCF7), Human Liver Hepatocellular Carcinoma (Hep3B) cancerous cells, African Green Monkey Kidney cells (Vero), and Human Amnion cells (FL). Methods: Chroococus minutus, Geitlerinema carotinosum, Nostoc linckia and Anabaena oryzae were collected from different freshwater habitats and identified. Each cyanobacterium was extracted with methanol. Antiproliferative activities of the cyanobacterial extracts were evaluated by [3-(4, 5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] (MTT) using HT29, C6, HeLa, A549, MCF7, Hep3B, Vero, and FL cell lines. Cytotoxicity was determined by lactate dehydrogenase (LDH) assay. Results: The cyanobacterial extracts showed varying antiproliferative and cytotoxic effects on cancer cells. G. carotinosum and N. linckia had significant inhibitory effect on C6 cell lines with half-maximal inhibitory (IC50) levels of 112.69 and 121.48 μg/mL, respectively. TGI values for G. carotinosum and N. linckia were 65.07 and 70.61 μg/mL, respectively. LC50 values for these two cyanobacteria were 386.64 and 760.55 μg/mL, respectively. In addition, cyanobacterium A. oryzae displayed excellent antiproliferative effect on MCF7 cancer cells (GI50 = 2.04 μg/mL). The extracts displayed the cytostatic effect on the cell lines. Conclusion: G. carotinosum, N. linckia and A. oryzae exhibit significant activity on various cancer cells. Hence these cyanobacteria may offer promise as anticancer agents.


INTRODUCTION
Cyanobacteria, known as cyanoprokaryotes or blue-green bacteria, and gram negative prokaryotes are some of the oldest living forms in the world, dating back to 3.7 billion years [1,2].The cyanobacteria have significant activities arising from production of some bioactive secondary metabolites [3].Filamentous sea cyanobacteria have been reported to prevent cancer growth, neurodegenerative and infectious diseases [4].Sea cyanobacteria include a great variety of compounds with pro-apoptotic effects [5].Sea cyanobacteria Synechocystis sp. and Trop J Pharm Res, November 2018; 17(11): 2184 Synechococcus sp. were reported to inhibit human promyeloctic leukemia (HL-60) cells [6].Rise of cancer incidence and limitations of existing drugs make it desirable to develop new anticancer molecules from various sources including freshwater cyanobacteria [7].It was reported that water extracts of Nostoc muscorum and Oscillatoria sp.showed high anticancer activity against Ehrlich Ascites Carcinoma cell and Human Hepatocellular (HepG2) cells [8].In addition, aqueous extract of Spirulina sp. have been shown to inhibit Hepatic Stellate cell (HSC).Besides, crude extracts of six cyanobacteria (Phormidium sp., Geitlerinema sp., Arthrospira sp., Phormidium sp.Phromidium sp. and Leptolyngbya sp.demonstrated concentrationdependent inhibitions of Homo Sapiens Kidney Carcinoma and Homo Sapiens Colon Colorectral Adenocarcinoma [9].Similarly, cyanobacterium Cyanothece sp. had high anticancer effect on extracellular T-lymphoma cells [10]. In the present study, anticancer properties of freshwater cyanobacteria species (Chroococcus minutus, Geitlerinema carotinosum, Nostoc linckia, Anabaena oryzae) were investigated on HeLa, HT29, A549, MCF7, Hep3B, C6 cancer cells and FL, Vero cells.

EXPERIMENTAL Isolation and culture of cyanobacteria
Cyanobacteria were collected from freshwater habitats around Tokat Yesilirmak River.Collected and identified cyanobacteria were isolated mechanically under an inverted microscope.The cyanobacteria C. minutus and G. carotinosum were grown in a 250 ml Erlenmeyer consisting of Bristol medium (125 mL).N. linckia and A. oryzae were cultured in an Erlenmeyer vessel containing 125 ml BG-11 medium [11].The cells in the vessel were incubated under fluorescent lamp at 26 ± 2 °C for 15 days.Then, cyanobacteria were centrifuged and dried at 60 °C.

Cyanobacteria extraction
Each dry cyanobacterium was extracted with methanol (50 mL) for 48 h.The solvent was filtrated and then removed by rotary evaporator to yield the extract which was subsequently dissolved in dimethyl sulfoxide (DMSO) (1.5 mL) for storage at 4 °C until use [12].

Cell lines
HeLa, MCF-7, Hep3B, A549, C6, HT29 cancer cell lines, along with FL cell and Vero kidney epithelial cell were used.Cell lines were incubated on a medium (Dulbecco's Modified Eagle Medium) enriched with 10% fetal bovine serum and 2% penicillin Streptomycin at 37 °C and 5% CO 2 environment.Analyses were carried out after cells reached a certain density.

MTT cell proliferation assay
MTT test was used to evaluate the effects of extracts on cell proliferation [13].After exposing cancer cells to the extracts for 24 h, MTT test was performed.
A stock solution was prepared by mixing 5 mg/ml MTT solution with RPMI1640 medium (without phenol red).This new solution was added to the reaction medium with incubation for 4 h.Absorbance measurement was carried out by a spectrophotometer at 560 nm.

Calculation of GI 50 , TGI, LC 50 and IC 50 parameters
At the end of incubation, cell inhibition was calculated.
The half-maximal inhibitory concentration of the extracts (IC 50 ) and 5FU were calculated by XLfit5 software as in Eq 1 [14].
where A is the absorbance of the extracts and B absorbance of untreated cells.GI 50 (50 of % Growth inhibition), TGI 50 (Total growth inhibition) and LC 50 (50% of drug causing cell kill) parameters were calculated for the extracts using Eqs 2 and 3.

Statistical analysis
The experiments were performed in triplicate and all data are expressed as mean ± standard deviation (SD).The data were analyzed by oneway ANOVA using SPSS software (version 21.0).

Anticancer activity
GI 50 , TGI, LC 50 and IC 50 values of C. minutus and G. carotinosum were given in Table 1.The antiproliferative activity of N. linckia and A. oryzae are shown in Table 2. Anticancer drug 5FU was used as a positive control (Table 3).
Compared with IC 50 values of 5FU and cyanobacterial extracts, G. carotinosum and N. linckia showed better activity against C6 cell lines (112.69 ± 0.07 μg/mL and 121.48 ± 0.09 μg/mL, respectively) than 5FU did (134.67± 1.02 μg/mL).A comparison with the standard (5FU) showed that the extracts had considerable levels of antiproliferative effects with potential to be used in pharmacology.

Lactate dehydrogenase (LDH) release
The cytotoxic effects caused by extracts in the present study was determined by measuring LDH at various concentrations (15, 30, 60 and 120 µg/mL) (Figure 1).Cyanobacterial extracts showed low cytotoxicity at all concentrations on HeLa and A549 cell lines (~10 -20 % cytotoxicity).It has been found that at low concentrations, the cytotoxic effect of the extracts on MCF7 cell line was very low and increased along with the concentration (~5 -40 % cytotoxicity).As in MCF7 and the Hep3B cell line, the toxicity was low at low concentrations.The extracts were found to be more toxic on other cell lines (HT29, C6, FL). C. minutus and A. oryzae had low cytotoxic effects on Vero cell lines, but their cytotoxicities were high in other extracts.Compared to standard 5FU whose cytotoxicity is known to be 5 -15 %, the extracts were found to have cytostatic effects on HeLa, A549, MCF7 and Hep3B cells.Because of their low toxicity and high antiproliferative effects, the extracts examined in the present study are suitable for drug development and they merit more advanced pharmacological investigations.

Morphological correlation with cytotoxic activity
Morphological changes caused by high anticancer activity of the G. carotinosum extract on C6 cell and low cytotoxicity on the HeLa cell were examined by inverse phase-contrast microscopy (Figure 2). .Due to the increasing cancer incidence in the world and high level of side effects by commonly used chemotherapy drugs, there has been an interest in use of natural products in cancer treatment recently.In the present study, antiproliferation and cytotoxicity features of cyanobacteria were examined on various cell lines to reveal their anticancer potential.Of the cyanobacteria studied, G. carotinosum showed the highest anticancer activity.It has been reported that anticancer effects of single filament cyanobacteria are high [22].Similarly, Geitlerinema sp.CCC728 and Arthrospira sp.CCC729 were considered to be potent anticancer drug sources [9].In addition, the LC 50 is important in cancer research and the fact that the LC50 is low indicates the high activity of cancer cells [23].
LDH assay in culture medium is used as an indicator for cytotoxicity [24].Lactate dehydrogenase is a cytoplasmic enzyme found in all cells.When cells are exposed to toxic effects, plasma membrane integrity is impaired and LDH enzyme penetrates into cells.Thus, cell damage can be assessed by measuring LDH activity after exposure to toxic effects [25].In the present study, some of the cyanobacterial extracts displayed considerable antiproliferative activities on various cancer cell lines, and they did not have any cytotoxicity on FL (human amnion cells) and Vero (kidney epithelial cell) cells.Thus, these extracts have the potential to be used in pharmacology.

Figure 2 :
Figure 2: Effect of G. carotinosum extract on morphology of C6 and HeLa cells.DMSO-treated cells are controls DISCUSSION Cyanobacteria are oxygen producing organisms and they can live almost everywhere.They are a source of rich bioactive natural products [16].The medical value of cyanobacteria has been known for a long time.Indeed, some cyanobacteria have been used for the treatment of some illnesses such as gout, fistula and various types of cancer [17,18].Aqueous extract of Spirulina platensis was reported to inhibit growth of Human Pancreatic Adenocarcinoma (PA-TU-8902) [19].Similarly, Phormidium sp. was shown to inhibit the growth of HT29 and HeLa cells [20,21].