1 Department of Biochemistry, Faculty of Science, University of Allahabad, 211002 Allahabad, UP, India
2 Department of Biochemistry, College of Science, King Saud University, 11451 Riyadh, Saudi Arabia
3 Department of Zoology, Faculty of Science, Banaras Hindu University, 211005 Varanasi, UP, India
Academic Editor: Josef Jampílek
Abstract
Introduction: Natural phytochemicals are considered safe to use as
therapeutic agents. There is a growing trend toward exploring anticancer effects
of crude algal extracts or their active ingredients. Euglena tuba, a microalga,
contains excellent antioxidant potential. However, the anticancer property of E.
tuba has not been explored. This study investigates the chemical profiling as
well as antitumor property of methanolic extract of E. tuba (ETME) against
Dalton’s lymphoma (DL) cells. Materials and Methods: E. tuba, procured
from northern part of India, was extracted in 70% methanol, dried at room
temperature, and stored at –20
Keywords
- Euglena tuba
- Dalton's lymphoma cells
- anticancer activity
- cytotoxicity
- antiproliferative activity
- GC-MS
- phytochemicals
Cancer is a general term to define a number of diseases that are characterized by the uncontrolled proliferation of cells resulting from the altered signaling pathways, which regulate cellular functions [1, 2]. In 2020, the International Agency for Research on Cancer (IARC), published a report showing total number on new cancer cases to be about 19.3 million with a global cancer death of 10 million people (2020). This data is expected to be double by end of 2040 due to increasing practices of smoking, unhealthy diet, physical inactivity, and fewer childbirths countries [3]. According to an estimate, about 1.81 million new cancer cases and 0.96 million cancer deaths were reported in India during 2020. The birth/death ratio has been reported to be 2.4. Despite rapid advancement in the health care sector, cancer is still a major health issue. At present, cancer is treated using chemotherapy, radiotherapy and surgery [4].
Though chemotherapy is an effective method for cancer treatment, there is still urgent requirement to develop safe, cost effective and novel anticancer agents with higher efficiency [5]. The synthetic/conventional anticancer compounds used in the treatment have been, however, reported to exert severe toxicity to the patients mostly via alterations in the cellular redox balance and functions of mitochondria. The efficacy and cytotoxicity of many anticancer compounds have been tested under varying conditions of cell culture with different cancer cell lines [6].
Nonconventional therapeutic approaches to treat cancer involve the phytochemicals isolated from different traditional medicinal plants. The plant based chemical ingredients have been shown to possess numerous medicinal properties. They could be used as alternative medicines to cure cancer, diabetes, inflammation and microbial infections [7, 8]. The phytochemicals regulate various molecular pathways and can be employed to inhibit proliferation of cancer cells, to inactivate the carcinogens, to induce apoptosis via arresting cell cycle and to increase the immune and redox systems [9]. The phytochemicals and their derivatives have been identified as suitable candidates for anticancer drug development due to their pleiotropic actions on targets and specific actions on tumor cells without affecting normal cells [10]. The biological targets of phytochemicals were found to be involved in antiproliferative, antiangiogenic, antimetastatic, and proapoptotic effects in mammalian cells or the ability to reduce oxidative stress [11, 12]. The antioxidants and other phytochemicals contained in lower plants such as algae have been displayed to arrest carcinogenesis [13].
Euglena tuba (Carter) (Family-Euglenaceae) is a unicellular
euglenozoa distributed in most aquatic bodies all over India throughout the year.
The red coloured euglenozoa blooms from India were recognized as Euglena
tuba, E. haematodes and E. orientalis [14]. Different
Euglena species exhibit redox properties as it contains plenty of
antioxidant compounds like vitamins A, B, C and E,
Due to the presence of the relatively nontoxic compounds in the algal extracts, their applications in human medicines against HIV-1, pathogenic microbes and genomic mutations, and tumor are quite popular. These compounds have also been shown to act as strong immuno-stimulants and immune-potentiator [32]. The algal bioactive compounds have potential to neutralize reactive oxygen species (ROS). ROS overproduction may result in genomic instability and cellular damage, as well as carcinogenesis. The present chemotherapeutic anticancer drugs are known to exert severe side effects and hence limit their long-term applications. Recently, more attention is being paid towards the discovery of plant based novel anticancer agents from the marine algae [33].
The compounds isolated from natural sources for the treatment of cancer are expected to exert either no or only minimum side effects. The scientists worldwide are engaged in designing the targeted therapeutics that can specifically exterminate cancer cells without harming the normal and healthy cells.
In this research paper, the chemical and biological characterization of the ETME
has been done and its effect on various cellular properties such as cell
viability, anti-proliferation activity, nuclear morphology, mitochondrial
membrane potential (
Roswell Park Memorial Institute Medium (RPMI-1640) and the culture medium were
obtained from HiMedia, Mumbai, India. Fetal bovine serum (FBS) was procured from
Invitrogen, CA, USA. Rhodamine (RH-123) and 4,6-diamidino-2-phenylindole (DAPI)
were obtained from Sigma Chemical Co. (St. Louis, MO, USA). MTT was procured from
Sigma Aldrich, India. Anti-Bcl
Algal sample were collected in the Baroh area of village Dhalwara, pond name Masran-ka-Talab, in the month of October 2016, in district Kangra of the state of Himachal Pradesh [34]. For identification of sample, E. tuba were preserved in formalin 4% and observed under the light microscope. The authenticity of E. tuba was verified by Dr. Rakesh Kumar (Algologist) and the voucher specimen was retained in the departmental herbarium for future reference with Accession No. WRS/GCD/DBH-002. It has been identified as E. tuba Carter belonging to family Euglenaceae, in Department of Botany, Wazir Ram Singh Govt. College Dehri, District Kangra (H.P.).
Algal samples were cleaned thoroughly three times in sterile distilled water
currently and removed inappropriate materials adhered to it. Finally, it was
centrifuged at 1000
The ETME were evaluated for the presence of the phytochemical analysis by using the following standard qualitative methods as described [36]. The components analysed for phytochemicals were phenolics, flavonoids, carbohydrates, alkaloids and ascorbic acid.
The phenolic content was estimated by using 1 mL extract (ETME). It was mixed
with 1.5 mL FC reagent (previously diluted to 1000 fold with distilled water)
followed by addition of 0.06% Na₂CO₃ (1.5 mL) solution. After incubation at 22
Total flavonoid content was determined using 0.5 mL extract. It was added to 5%
NaNO
Carbohydrate content of the extract was quantified using 10 mg of ETME. It was
hydrolysed with 5 mL of 2.5N HCl. The mixture was diluted to 10 mL with distilled
water, centrifuged and supernatant was used and mixed with 4 mL anthrone reagent.
The mixture was incubated at water bath 95
Ascorbic acid content quantification was accomplished according to an elucidated
technique [36]. In brief 1 mL aliquots of ETME (1 mg/mL) in water were mixed with
1 mL of 2,4-dinitro-phenylhydrazine reagent and incubated at 95
Alkaloid content was quantified using 3 mL ETME extract. It was mixed with 0.3
mL of FeCl₃ (2.5 mM FeCl₃ in 0.5 MHCl) followed by addition of (0.3 mL, 0.05 M 1,10
phenanthroline). After incubation for 30 min at 70
The ETME was analysed using a Thermo Scientific Gas
Chromatography-Traceultraver: 1100 and Mass Spectrometry- TSQ Duo. The oven
temperature was maintained at 220
The retention index of the compounds was identified by comparing the retention times and identification of each component was confirmed by the comparison of its retention index with data in the NIST library. Interpretation of Mass-Spectrum was carried out by using the database of the National Institute Standard and Technology (NIST) having more than 62,000 patterns. Spectrum of the known compound which are stored in NIST library was used to compare the spectrum of unknown component. The molecular weight, name, chemical structure and molecular formula of the components of the test materials were ascertained.
For the evaluation of the ETME mediated anti-tumor response in Dalton’s
lymphoma, the inbred populations of BALB/c (H2d) strain of mice of either sex at
8–12 weeks of age were used. In conventional cages (2 animals in each cage)
BALB/c (H2d) strain of mice received sterilized food and water ad
libitum. All animals were kept and maintained with utmost care under the
guidelines of the Institutional Animal Ethics Committee with CPCSEA Registration
No. 839/GO/Re/04/CPCSEA, dated 30/08/2017, University of Allahabad, Allahabad,
India. There is no any need to use euthanasia of animals in our experiment
because we collected the DL cells from DL bearing mice and injected into the
healthy mice of either sex at 8–12 weeks of age intraperitoneally (1.0
Evaluation of cytotoxicity of the extracts on DL cells was determined by the MTT
assay. This test is based on MTT (3-(4,5- dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide), which is reduced by the living cells to a
purple-blue precipitate of insoluble formazan. DL cells were harvested in RPMI
1640 medium (10% FBS and antibiotics solution) and seeded in 96 well culture
plate at cellular density of 2
From the mice bearing DL, the peritoneal DL cells were harvested. The
nonadherent cells were collected and washed three times with chilled PBS. The DL
cells (1
The morphological evidence of apoptosis was detected by the treatment of DL
cells (1
The changes in the mitochondrial membrane potential due to apoptosis induced
condition were studied by flow cytometry using rhodamine-123 as described earlier
[39]. The non-adherent cells were collected and washed twice with chilled PBS.
The DL cells count (1
The DL cells (1
The data was statistically analyzed by using one way analysis of variance
(ANOVA) with Tuckey post hoc test by Graph Pad Prism software (9, GraphPad
Software, CA, USA). p value
In the present study, the ETME revealed that the presence of various bioactive
phytoconstituents could be responsible for the therapeutic ability of ETME. The
results obtained from the colorimetric analysis of the ETME demonstrated the
presence of chemical components such as alkaloids, carbohydrates, flavonoids,
phenols and ascorbic acid. The ETME analysis showed presence of phenolic contents
to a significant level, i.e., 0.94
It was found that the ETME contained highest amount of alkaloid, with considerable amounts of flavonoid, ascorbic acid, carbohydrates and very low amount of phenolic content. In this study, preliminary phytochemicals analysis was not sufficient to evaluate the content of secondary metabolites present in ETME. Therefore, the quantitative phytochemical analysis was carried out using a methanolic solvent system of the ETME.
In the present study, we have reported the presence of some of the important
compounds in ETME as resolved by GC-MS analysis and also searched their
biological activities from previous studies. The GC–MS chromatogram of ETME
recorded a total of 631 peaks corresponding to different bioactive compounds that
were recognized by relating their peak retention time, peak area (%), height
(%) and mass spectral fragmentation patterns to that of the known compounds
described by the National Institute of Standards and Technology (NIST) library.
Out of 631 peaks, the major peaks based on the percentage area of peaks
The phytoconstituents in the ETME extract were found to be Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15hexadecamethyl; Hexasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl; Propyl-1-pentanol; 1-Hexanol,2-ethyl; Cyclohexasiloxane, dodecamethyl; Dimethyl phthalate; Dodecanoic acid, methyl ester; 2,4-Di-tert-butylphenol; Cyclotridecane; 1-Hexadecanol; Methyl tetradecanoate; 1-Hexadecanol; Methyl 13-methyltetradecanoate; Phthalic acid, butyl undecyl ester; Methyl 4,7,10,13-hexadecatetraenoate; Hexadecanoic acid, methyl ester; Methyl 2-ethylhexyl phthalate; 10-Octadecenoic acid, methylester; 9,12,15-Octadecatrienoic acid, methyl ester; Methyl stearate; 5,8,11,14-Eicosatetraenoic acid, methyl ester; cis-5,8,11,14,17-Eicosapentaenoic acid; Eicosane; Phthalic acid, di(2-propylpentyl) ester. The GC-MS chromatogram of ETME based on their retention time (RT) displays presence of 10 peaks as demonstrated in Fig. 1. The structure and biological functions of the some bioactive compounds present in the ETME are displayed in Table 1 (Ref. [41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63]).
Fig. 1.GC-MS chromatogram of ETME. RT signifies retention time of the peaks. The relative abundance of peaks is shown; maximum being at RT value of 36.00.
| S.N. | Compound name | Molecular weight | Molecular formula | % Area | Retention time | Biological activity | Reference |
| 1 | 
|
578 | C |
0.55 | 4.05 | Antimicrobial | [41] |
| OCTASILOXANE, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-HEXADECAMETHYL- | |||||||
| 2 | 
|
430 | C |
0.12 | 5.18 | Anti-microbial, antibacterial, anti-septic, hair conditioning agent, emollient | [42, 43, 44] |
| HEXASILOXANE, 1,1,3,3,5,5,7,7,9,9,11,11-DODECAMETHYL | |||||||
| 3 | 
|
130 | C |
6.94 | 5.45 | Not reported | – |
| PROPYL-1-PENTANOL | |||||||
| 4 | 
|
130 | C |
0.86 | 5.88 | Not reported | – |
| 1-HEXANOL, 2-ETHYL- | |||||||
| 5 | 
|
444 | C |
0.1 | 12.43 | Antifungal properties | [45] |
| CYCLOHEXASILOXANE, DODECAMETHYL- | |||||||
| 6 | 
|
194 | C |
1.96 | 16.06 | Not reported | – |
| DIMETHYL PHTHALATE | |||||||
| 7 | 
|
214 | C |
0.22 | 17.08 | Antibacterial, antiviral, antifungal | [46] |
| DODECANOIC ACID, METHYL ESTER | |||||||
| 8 | 
|
206 | C |
0.24 | 17.26 | Antibacterial, antiviral, antifungal activities | [47, 48, 49] |
| 2,4-DI-TERT-BUTYLPHENOL | |||||||
| 9 | 
|
182 | C |
0.22 | 18.29 | Not reported | – |
| CYCLOTRIDECANE | |||||||
| 10 | 
|
242 | C |
3.18 | 20.46 | anticancer, anti-inflammatory and antimicrobial, antioxidant activities | [50] |
| 1-HEXADECANOL | |||||||
| 11 | 
|
242 | C |
0.63 | 21.41 | plant metabolite, a flavouring agent and a fragrance | [51] |
| METHYL TETRADECANOATE | |||||||
| 12 | 
|
256 | C |
0.17 | 23.44 | Antioxidant, cancer-preventive, hypercholesterolemi, lubricant, nematicide | [52] |
| METHYL 13-METHYLTETRADECANOATE | |||||||
| 13 | 
|
376 | C |
0.08 | 24.69 | Antimicrobial, antibacterial, anti-inflamatory activity | [53] |
| PHTHALIC ACID, BUTYL UNDECYL ESTER | |||||||
| 14 | 
|
262 | C |
0.73 | 24.8 | Antioxidant, antibacterial activity | [54] |
| METHYL 4,7,10,13-HEXADECATETRAENOATE | |||||||
| 15 | 
|
270 | C |
6.98 | 25.36 | Antioxidant, antimicrobial, hemolytic, hemolytic, 5-alpha reductase inhibitor cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries | [50] |
| HEXADECANOIC ACID, METHYL ESTER | |||||||
| 16 | 
|
92 | C |
21.95 | 27.83 | Antimicrobial and Cytotoxic Activity | [55] |
| METHYL 2-ETHYLHEXYL PHTHALATE | |||||||
| 17 | 
|
296 | C |
0.55 | 28.57 | Antimicrobial potential | [56] |
| 10-OCTADECENOIC ACID, METHYL ESTER | |||||||
| 18 | 
|
292 | C |
0.38 | 28.69 | Anti-inflammatory and anti-arthritic | [57, 58] |
| 9,12,15-OCTADECATRIENOIC ACID, METHYL ESTER | |||||||
| 19 | 
|
298 | C |
3.65 | 28.96 | Antidiarrheal, cytotoxic and antiproliferative activities | [59, 60] |
| METHYL STEARATE | |||||||
| 20 | 
|
318 | C |
0.17 | 31.39 | Antifungal, antibacterial, antitumor cytotoxic effects | [61] |
| 5,8,11,14-EICOSATETRAENOIC ACID, METHYL ESTER | |||||||
| 21 | 
|
302 | C |
0.18 | 31.56 | Not reported | – |
| CIS-5,8,11,14,17-EICOSAPENTAENOIC ACID | |||||||
| 22 | 
|
282 | C |
0.11 | 33.12 | Antifungal compound | [62, 63] |
| EICOSANE | |||||||
| 23 | 
|
390 | C |
0.08 | 35.28 | Anticancer activity | [55] |
| PHTHALIC ACID, DI(2-PROPYLPENTYL) ESTER |
The phytochemical and bioactive molecules present in ETME were Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl; Hexasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl; Cyclohexasiloxane, dodecamethyl; Dodecanoic acid, methyl ester; 2,4-Di-tert-butylphenol; Phthalic acid, butyl undecyl ester; 10-Octadecenoic acid, methyl ester; Eicosane. The bioactive molecules such as 1Hexadecanol; Methyl 13-methyltetradecanoate; Methyl 4,7,10,13- hexadecatetraenoate; Hexadecanoic acid, methyl ester; Methyl 2-ethylhexyl phthalate; Methyl stearate; Phthalic acid, di(2-propylpentyl) ester is reported as anticancer, anti-inflammatory and antimicrobial, antioxidant activities. Another compound 9,12,15-Octadecatrienoic acid, methyl ester has been shown to exhibit the anti-inflammatory and anti-arthritic property and Methyl tetradecanoate compound show the plant metabolite, as a flavouring agent and a fragrance. The library search report and GC-MS chromatogram of ETME are displayed in Fig. 1.
In the in vitro condition, the treatment with ETME may induce the death
of DL cells. In order to assess the cytotoxic activity of ETME on DL cells, MTT
assay was performed as described in Materials and Methods. The data presented in
Fig. 1 indicated that ETME exhibited anticancer potential to markedly inhibit the
growth of DL cells. After the incubation periods of 24 h (Fig. 2A) and 48 h (Fig. 2B), the percentage cell death was calculated. The results indicated that ETME
significantly inhibited the proliferation of DL cells in the concentration
dependent manner (**p
Fig. 2.Effect of ETME on viability and growth of DL cells. MTT assay
of DL cells treated with different concentrations of ETME (25, 50, 100, 200, 250
We studied the mode of death of DL cells upon treatment with ETME by observing
the nuclear morphology using DAPI staining method as described above. The results
reflected that the nuclear abrogation of DL cells in the ETME (200
Fig. 3.Effect of ETME on nuclear morphology of DL cells as observed by
DAPI staining. DL cells were treated with ETME (200
The cellular morphological changes in DL cells upon treatment with ETME were
also monitored by acridine orange/ethidium bromide (AO/Eb) staining as described
in Materials and Methods. The treatment of DL cells with ETME revealed changes
associated with apoptosis as indicated by the arrows. In both the groups viz.
control and the treated DL cells, appearance of green fluorescence with intact
green nucleus represents live cells. Morphological changes such as membrane
blebbing, cell shrinkage and a number of red/orange cells (apoptotic cells) were
observed in DL cells on treatment with ETME (200
Fig. 4.Composite images of acridine orange/ethidium bromide (AO/EtBr)
stained DL cells after treatment with ETME. The yellow and green arrows
represent apoptotic and live cells, respectively. DL cells were treated with ETME
(200
After treatment with ETME, the status of mitochondrial membrane potential
(
Fig. 5.Effect of ETME on the mitochondrial membrane potential
(
After observing sharp decrease in the mitochondrial membrane potential into DL
cells due to ETME treatment, the expression of some key proteins involved in the
regulation of apoptosis of DL cells by Western blot analysis were further
examined. The results presented in Fig. 6 indicated an increase in pro-apoptotic
protein, Bax, which may contribute to the release of cytochrome C (Cyt-c) from
mitochondria and activation of intrinsic apoptotic pathway. The results shown in
Fig. 6 indicated that ETME was able to down-regulate the expression of
antiapoptotic protein Bcl
Fig. 6.Effect of ETME on the levels of expression of apoptosis related
protein. Western blotting was carried to decipher the effect of ETME on the
expression of apoptotic proteins viz. p53, Bcl
The analysis and extraction of plant material plays a vital role in the growth, development, upgrading, and quality control of herbal formulations [64]. The study of medicinal plants helps to explore their therapeutic potential to cure the humans and animals from various diseases. The current investigations on ETME revealed the presence of various phytoconstituents. These bioactive phytoconstituents could be responsible for the therapeutic ability of various extracts of E. tuba. The microalgae possess numerous metabolites [65, 66, 67, 68] responsible for their varied physiological activities with biomedical significance. Flavonoids and phenolic compounds are known to contain antioxidant, anti-inflammatory, anti-cancer, anti-diabetic, and anti-pypertic activities [68, 69, 70, 71, 72, 73].
Alkaloids extracted from plants show antimicrobial [74], antitumor and antiviral activities [75, 76]. Similar to our results, recently some phytochemicals in ETME such as steroids, alkaloids, phenols, flavonoids, saponins, tannins, anthro-quinone have been reported to contain potential antibacterial, anticancer properties [77]. The results from the study conducted by others workers have also shown the presence of flavonoids, carbohydrates, tannins, phenols, amino acids, alkaloids, steroids, proteins, terpenoids, phytosterols, saponins and diterpenes in E. tuba, which could be responsible for its therapeutic ability [78].
The GC–MS analysis of ETME revealed the presence of 23 phytochemical compounds, which could contribute to the medicinal properties of this plant species [79, 80, 81]. The analysis also reported that the compound Octasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl has the antimicrobial activity [41]. The compound Hexasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl possesses the anti-microbial, antibacterial, anti-septic, hair conditioning agent, emollient [42, 43, 44]. The Cyclohexasiloxane, dodecamethyl is known to exhibit antifungal properties [45]. The Dodecanoic acid, methyl ester was found to have antibacterial, antiviral, antifungal activity [46]. The 2,4-Di-tert-butylphenol or 2,4-bis (1,1-dimethylethyl)-phenol (2,4-DTBP), contained the antibacterial, antiviral, antifungal activities [47, 48, 49], while 1-Hexadecanol present in ETME exhibiting anticancer, anti-inflammatory, antimicrobial and antioxidant activities [50]. The compound, methyl tetradecanoate, acts as a flavouring and fragrance agent [51]. The compound Methyl 13-methyltetradecanoate had antioxidant, cancer-preventive, hypercholesterolemia, lubricant, nematicide properties [52]. Other bioactive compounds such as phthalic acid, butyl undecyl ester have been documented to contain a broad spectrum of antimicrobial, antibacterial, anti-inflammatory activities [53]. However, Methyl 4,7,10,13-hexadecatetraenoate was found to possess characteristic of antioxidant and antibacterial activity [54].
Ouyang et al. [50] have reported the hexadecanoic acid, and its methyl ester with the antioxidant, antimicrobial, and hemolytic functions. These molecules also acted as 5-alpha reductase inhibitor (cancer enzyme). The methyl 2-ethylhexyl phthalate, has been reported to be capable of exhibiting high antimicrobial and cytotoxic properties [55]. The compound,10-octadecenoic acid, and its methyl ester are reported to act as antimicrobial agents [56]. Another compound 9,12,15-Octadecatrienoic acid, methyl ester has been shown to exhibit the anti-inflammatory and anti-arthritic property [57, 58]. The Methyl stearate compound has been suggested to contain potential bioactivities, such as antidiarrheal, cytotoxic and antiproliferative activities [59, 60]. The compound 5,8,11,14-Eicosatetraenoic acid, methyl ester has antifungal, antibacterial, antitumor cytotoxic effects [61]. Some workers have shown that the compound Eicosane possessed the antifungal property [62, 63]. Phthalic acid, di (2-propylpentyl) ester from ETME was found to be capable of producing high multi therapeutic properties such as anticancer activity [55]. Out of 23 compounds present in ETME only 6 compounds such as 1-Hexadecanol; Methyl 13-methyltetradecanoate; Methyl 2-ethylhexyl phthalate; Methyl stearate; 5,8,11,14-Eicosatetraenoic acid; Phthalic acid, di(2-) ester were reported to contain anticancer property. There was no evidence found for the presence of any biological activity of the compounds such as Propyl-1-pentanol; 1-Hexanol, 2-ethyl; Dimethyl phthalate; Cyclotridecane and cis-5,8,11,14,17-Eicosapentaenoic acid.
Cancer being a very serious health problem represents the second most threatening disease in humans after cardiac complications [82]. The cancer related research has always been paid a great attention, particularly in the direction of design and development of effective and safe drugs for its chemotherapy, specially derived from plants in journal and microalgae in particular [83]. Different Genus such as Chlorella, Cladophoropsis, Codium, Dunaliella, Enteromorpha, Helimenda, Udocea, and Ulva were previously reported to be able to produce high value compounds (HVC) with potential bioactive property against cancer [82, 84, 85, 86, 87, 88]. These findings have suggested the need of further investigation of algal species inhabiting in both the marine and sweet water to explore active natural molecules to complement the existing anticancer medicines with very small and no toxicity. In order to address the complexity of cancer, some novel bioactive compounds with multipronged efficacy are urgently required to be investigated [89].
In this study, ETME has been screened for its probable anticancer activity using cell culture system involving DL cells. It was observed that ETME was able to inhibit the growth of DL cells through the induction of apoptosis. However, the cytotoxic effects of an algae, A. armata, in cell lines have been reported by Zubia et al. [90] against the human cervical cancer cell line, i.e., HeLa. The methanolic extracts of two green algae, E. intestinalis and R. riparium exhibited antiproliferative activity [91]. The methanolic fraction of another microalgae, S. triplicate, was observed to be highly active against the progression of human lung cancer cells (A549). Further, the extracts of other microalgae namely H. cornea and G. longissima were found to elicit the cytotoxic effects on RAW264.7 cells at low concentration as measured by the MTT assay; its anticancer potential being associated to the presence of high amount of many halogenated phenolic compounds [92]. These compounds have been reported for their cytotoxic potential against some cancer cell lines [93]. The aqueous extract of Gracilaria corticate, a microalgae, have been evaluated by some workers for their antitumor activities in the human leukemic cell lines such as Jurkat and molt-4 cells and their results indicated that these extracts were able to significantly reduce the tumor cells proliferation [94].
The anticancer agents are known to possess the characteristic to induce apoptosis in cancer cells, though various other mechanisms have also been reported by which different molecules cause attenuation of tumor angiogenesis [95] including induction of apoptosis or necrosis or immune- stimulation [96]. The mechanisms by which algae bioactive function mainly rely on to their distinct chemical constituents and biological properties [97] have demonstrated that fucoidan (a complex polysaccharide found in many species of brown seaweed functions against the proliferation of lung cancer through causing delay in the development of cancer, elimination of cancer cells, and display of the synergistic effect with the anticancer chemotherapeutics.
The characteristic feature of apoptosis includes altered cellular and nuclear
morphology, as well as reduction in the mitochondrial potential. The bioactive
compounds present in the ETME have been reported to possess antimicrobial,
antiviral, and antioxidant properties [98]. The results from the present study
have indicated that ETME (200
The decrease in the mitochondrial membrane potential (
The expression levels of the proapoptotic Bax and antiapoptotic Bcl
It is known that the transmission of the apoptotic signals to the apoptotic regulatory system of a living cell takes place primarily through two pathways known as extrinsic and intrinsic. In addition, it also involves perforin/granzyme pathway [110]. The “extrinsic” pathway involves the death receptors which bind their specific ligands. The receptor-ligand binding undergoes conformational changes and causes activation of the caspase cascade [111]. This event may be responsible for inducing the release of mitochondrial cytochrome C into the cytoplasm, thereby leading to the activation of effector caspases. In contrast, “intrinsic” pathway is directly triggered by numerous apoptotic signals that facilitate cytochrome C release [111, 112]. The release of Cyt-c in cytosol further activated caspase dependent apoptosis. In general, apoptotic pathway consists of sequential biochemical events including the activation of caspases as described [113], a family of cysteine proteases that plays crucial roles in apoptotic signaling.
The tumor suppressor protein p53 has been shown to play a key role in the
regulation of apoptosis and cell cycle [113]. In addition, p53, a tumor
suppressor gene, functions as a negative regulator of Bcl
The results obtained involving in vitro studies with DL cells
demonstrated ETME mediated morphological changes in the cells such as nuclear
fragmentation, migration of nucleus and chromatin condensation associated to
apoptosis. The cytotoxicity of ETME in DL cells, however, exhibited a positive
correlation with time, i.e., ETME exerts more anticancer effect at higher time of
exposure duration. The ETME treatment also caused an increase in the expression
of the proapoptotic protein Bax, which would have contributed to the release of
cytochrome C (Cyt-c) from mitochondria into cytosol causing marked reduction in
the mitochondrial membrane potential and activation of intrinsic apoptotic
pathway. The ETME was able to cause down-regulation of the expression of
antiapoptotic protein Bcl
SPG collected E. tuba and prepared the methanolic extract, generated the DL induced ascites in the Balb/c mice, cultured the cells and conducted the required experiments and wrote the primary manuscript. RKS, PKV, SK planed the experiments, and statistically analysed the results. NJS, HAK, SHA and ASA organized the contents, removed the plags and edited the manuscript. AA and BS examined the display of results in tables and figures and prepared the final draft of the manuscript.
The approval of the Institutional Animal Ethics Committee was obtained with CPCSEA Registration No. 839/GO/Re/04/CPCSEA, dated 30/08/2017, University of Allahabad, Allahabad, India.
SPG acknowledges the University Grants Commission-New Delhi for providing financial support in the form of a research fellowship.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding the Research Group Grant No. RGP-1435-066.
The authors declare no conflict of interest.