Skin mucus extract derived from Channa punctatus, Channa striatus, and Heteropneustes fossilis induces apoptosis and suppresses proliferation in human adenocarcinoma cells via ROS mediated pathway and cell cycle arrest

doi:10.21203/rs.3.rs-4704912/v1

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Research Article

Skin mucus extract derived from Channa punctatus, Channa striatus, and Heteropneustes fossilis induces apoptosis and suppresses proliferation in human adenocarcinoma cells via ROS mediated pathway and cell cycle arrest

https://doi.org/10.21203/rs.3.rs-4704912/v1

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Purpose

Skin mucus of freshwater fish species living in muddy habitats possesses the ability to inhibit the growth of human pathogens, and may also have the potential to inhibit the growth of cancer cells. This study first explored the anti-cancer activity of skin mucus acetic extracts of Channa punctatus (CPMA), Channa striatus (CSMA) and Heteropneustes fossilis (HFMA) against lung adenocarcinoma.

Methods

The cytotoxicity of the extract(s) on A549 and NRK-52E cells were evaluated using MTT assay and morpholological analysis of cells. Nuclear condensation, ROS generation, apoptosis and cell cycle analysis was performed on A549 cells. The protein profiles of extract(s) were evaluated via. SDS-PAGE.

Results

SDS-PAGE showed 15.2–69.1, 14.3–70.2 and 14- 97.5 kDa proteins in CPMA, CSMA and HFMA respectively. The extracts inhibited cellular proliferation in A549 cells in a dose-dependent manner, with IC50 values of 619 (CPMA), 591 (CSMA), and 538 µg/mL (HFMA). Morphological changes in the cells, presence of condensed nuclei and intracellular production of ROS increased dose dependently in the order HFMA > CSMA > CSMA. Early and late apoptotic cells were observed at low and high doses respectively. Moreover, G₂/M arrest was found at low dose and apoptosis at high dose of CPMA and CSMA, while both G₂/M arrest and apoptosis was found in a dose dependent manner in HFMA treated cells. Interestingly, no toxicity was observed against healthy NRK-52E cells.

Conclusion

HFMA possess the highest cytotoxic effect against A549 cells, followed by CSMA and then CPMA, which directly corresponds to the range of MW of proteins present therein.

Fish skin mucus

lung cancer

apoptosis

freshwater fish

cell cycle.

Aquatic organisms comprise about half of the entire biodiversity prevailing in the world [1] Researchers have increasingly turned towards this relatively unexplored natural source for identification and isolation of natural products for potential treatment and prevention of several diseases including cancer [2–5]. Among the aquatic fauna, fishes are the most diverse and highly specialized group of animals, which evolved to thrive in micro-organism rich environment. Therefore, they highly rely on their innate immunity for protection against invading pathogens and other diseases. The immediate innate immunity in fishes is provided by the epidermal mucus, since the other immune mechanisms are slow because of their poikilothermic nature [6, 7]. The fish skin mucus (FSM) mucus is a slimy secretion consisting of proteins, antibacterial enzymes, mucopolysaccharides and water [8]. Its bioactive components constituting the innate immunity are lectins, glycoproteins, proteolytic enzymes, C-reactive protein, lysozyme, flavoenzymes, antimicrobial peptides, complement proteins and other immunoglobulins [9, 10]. FSM is a multifunctional by-product, which is considered as a unique source of protein carrying a range of pharmacological properties such as antibacterial [2], anti-proliferative [11] and cytotoxic [12]. Its potential for treatment of human diseases leads to increase in research for application in human medicines.

A number of studies have been reported on the antimicrobial activities of FSM derived from freshwater species [13–19], but exploration of their anticancer activity is in its growing phase [11, 20–23]. Among the various type of cancers, lung cancer continues to be a significant global health challenge, eliciting countless fatalities in both men and women [24, 25]. Over 2.5 million cases of lung cancer are diagnosed annually, exceeding 1.8 million deaths worldwide because of the poor prognosis and high expenditure [26]. The current medical interventions include chemotherapy and radiotherapy which are harmful to healthy cells along with cancer cells leading to long-term side effects and unfavourable clinical outcomes. The inadequacy of cancer treatment regimens to completely eradicate the disease has paved the way for search of alternative natural products with selective cytotoxic effects against malignant cells and little to no effects on normal, healthy cells. Bioactive proteins/peptides from several aquatic animals like urochordates, poriferans, ascidians, molluscs and fishes have gained recent attention on account of their promising anticancer potential [27–30].

Freshwater fish species inhabiting muddy habitats such as channidae, chariidae and siluroidea have high levels of innate immune factors for their survival [30] and therefore, can be a good candidate to be explored for its antimicrobial and anticancer potential. To date no research has been conducted to explore the anticancer potency of Channa punctatus (Bloch, 1793) and Channa striatus (Bloch, 1793), commonly known as murrels; and Hteropneustes fossilis (Bloch, 1794) commonly known as stinging fish. These fishes belong to the family channidae and siluroidea respectively and are quite tolerant of turbid conditions and low oxygen levels (possess accessory respiratory organs). These indigenous species are economically and traditionally important for medicinal purposes [32–34]. Strong antibacterial activity has been reported in the acetic extracts of C. punctatus and C. striatus skin mucus [13, 35] while skin mucus of H. fossilis is reported to have bactericidal as well as antifungal properties [36, 37]. Our previous findings have reported that C. punctatus, C. striatus and C. gariepinus possess antibacterial activity against various gram-positive and gram-negative bacteria. Three types of extracts (crude, acetic and organic) were prepared of which the highest antibacterial activity was recorded in the acetic extract(s) [2]. Several other researchers have also reported the acetic extract of FSM to be more effective in killing human pathogens as compared to crude, organic and aqueous extracts [13, 38]. Therefore, in the present study acetic extract(s) of skin mucus from C. punctatus, C. striatus and H. fossilis were selected for the exploration of anti-proliferative and apoptotic potential against lung adenocarcinoma cell line (A549). The protein content of the acetic extract(s) was quantified and molecular weights were estimated via. SDS-PAGE. The anticancer activity was evaluated using range of established in vitro techniques.

Sample collection and preparation

The samples of C.punctatus, C.striatus and H.fossilis were collected from river Gomti at Lucknow with the assistance of a local fisherman. To ensure accurate identification, keys and manuals provided by Talwar and Jhingran [39] and Jayaram [40] were followed. After 15-day acclimatization period in aquarium under standard laboratory conditions, the skin mucus was extracted from the surface of ten healthy specimens, weighing between 250-350g using a sterile scrapper. The extracted mucus was subsequently lyophilized (LyoQuest, Telstar, Japan) and stored at -20°C. Preparation of acetic extract(s) of fish mucus was done following method of Diamond et al. [41] with some minor modifications. Briefly, 10mg of lyophilized mucus sample was mixed with 1mL of 10% acetic acid (w/v) and subjected to boiling water bath for 5 min. The mixture was subsequently cooled on ice and centrifuged at 10,000 rpm for 35 minutes at 4°C. The resulting supernatant was collected and stored at -20°C. The extract(s), referred to as CPMA (C. punctatus skin mucus acetic extract), CSMA (C. striatus skin mucus acetic extract) and HFMA (H. fossilis skin mucus acetic extract), were further utilized as such for in-vitro studies.

Protein estimation and SDS-PAGE analysis

The protein content in fish skin mucus acetic extract was quantified by the method of LOWRY [42] using bovine serum albumin (BSA; Sigma-Aldrich) as standard. The approximate molecular mass range of proteins present in the three fish skin mucus acetic extract(s) were analyzed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli [43] with slight modifications. Briefly, mucus extract(s) were mixed with sample buffer in a ratio of 4:1 (v/v). SDS-PAGE was carried out on a discontinuous gel system comprising of 5% stacking gel (pH 6.8) and 12% resolving gel (pH 8.8), followed by staining and destaining of the gel. The gels were visualized, documented and preserved.

Cell culture

Human adenocarcinoma cell line (A549; RRID: CVCL_UJ49) and non-cancer rat kidney epithelial cell line (NRK-52E; RRID: CVCL_0468) were acquired from the cell repository of National Centre for Cell Sciences (NCCS), Pune, India and were used at low passage numbers (< 20). A549 is human alveolar type II epithelial cell line and NRK-52E is rat renal tubular epithelial cell line. The cells were cultured in dulbecco’s modified eagle medium nutrient mixture F-12 (DMEM/F-12; Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% heat inactivated foetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and antibiotic/antimycotic solution (10,000 U of penicillin and 100 µg/mL of streptomycin; HiMedia, Mumbai, MH, India). The cell cultures were maintained in a humidified incubator at 37°C under 5% CO₂ (Eppendorf, New Brunswick, USA).

Cell viability assay

Cell viability of extract(s) treated A549 and NRK-52E cells was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay [44]. Briefly, A549 cells and NRK-52E were seeded at a density of 1 × 10⁵ cells/well in 96 well plates for 24 h and treated with 400–800 µg/mL CPMA, CSMA and HFMA for A549 cells and 400–900 µg/mL CPMA, CSMA and HFMA for NRK-52E cells. After 48 h of treatment, both cell lines were incubated at 37°C with 20 µL of MTT dye (5mg/mL; HiMedia) for 3 h and the plates were read at 545 nm in an ELISA plate reader (Biorad-PW41, Bio-Rad Laboratories Inc., Hercules, CA, USA) after adding 200 µL DMSO (Merck Millipore, Darmstadt, Germany) to each well to dissolve formazan crystals.

Morphological examination

The morphology of treated and untreated A549 and NRK-52E cells were analyzed under an inverted phase contrast microscope (Nikon Eclipse TS100, Nikon corp., TYO, Japan) after treating A549 cells with CPMA and CSMA in the range 500–600 µg/mL in separate experiments versus treatment of NRK-52E cells with CPMA, CSMA and HFMA in the range 500–900 µg/mL for 48 h. On the other hand, A549 cells were treated with HFMA in the range of 400–600 µg/mL for 48 h.

DAPI staining

4′6-diamidino-2-phenylindole (DAPI) is a blue-fluorescent DNA dye that enhances the fluorescence intensity of DNA by ~ 20-fold when bound to AT regions of ds-DNA. Nuclear condensation/fragmentation in the A549 cells was assayed using DAPI dye (Sigma-Aldrich Co., St. Louis, MO, USA) as reported by Ahamad et al. [45] with some minor modifications. Briefly, A549 cells were seeded at a density of 5 × 10⁴ cells/well in a 24-well plate for 24 h and treated with 500–600 µg/mL of CPMA and CSMA and 400–600 µg/mL of HFMA for 48 h in separate experiments. Cells were washed and fixed in ice-cold methanol (Merck, Darmstadt, Germany) for 10 min. Fixed cells were stained with 10 µM DAPI dye and observed under inverted fluorescence microscope (Zeiss AxioVert 135, Carl Zeiss, Oberkochen, Germany). For each treatment, cells were quantified in triplicate and percent apoptotic cells were calculated by counting 100 cells per sample in different fields using fluorescence microscope.

ROS generation assay

ROS generation by certain drugs induces apoptotic cell death in cancer cells. Hence, ROS generation was detected by the method of Jafri et al. [46] using 2′, 7′-dichlorodihydrofluorescein-diacetate dye (DCFH-DA; Sigma-Aldrich) with some modifications. Briefly, A549 cells were seeded and treated in separate experiments as described above. The cells were washed and incubated in dark with 10 µM DCFH-DA at RT for 30 min. Reaction mixture was aspirated and cells were washed with phosphate buffer Saline (1X PBS; HiMedia) and viewed under fluorescence microscope (Zeiss AxioVert 135). Images were quantified using image J software 1.2.4 (RRID: SCR_003070; Bioimage XD and Cell Profiler, imajej.nih.gov/ij/). Results were calculated as percentage of fluorescence intensity with respect to untreated well (control).

AO-EtBr dual staining

The morphology of apoptotic cells was analyzed using acridine orange-ethidium bromide (AO-EtBr; HiMedia) according to the method of Xin et al. [47]. Briefly, A549 cells were seeded in 24-well cell culture plates in separate experiments and treated as mentioned above. The cells were stained with 2.5 µg/mL of AO-EtBr in the ratio of 1:1 and incubated in CO₂ incubator for 10 min. Subsequently, cells were washed with 1X PBS and photographed under fluorescence microscope (Zeiss AxioVert 135).

Cell cycle analysis

Cellular DNA content analysis of A549 cells via cell cycle phase distribution was performed using flow cytometer.⁴⁸ Briefly, in separate experiments, 1 × 10⁶ cells/well were seeded in 6-well plates for 24 h and then treated with CPMA and CSMA at 500 and 700 µg/mL and HFMA at 400 and 600 µg/mL. After 48 h incubation, the cells were harvested by trypsinization, washed in 1X PBS (ice-cold) and fixed in 70% ethanol (Merck) for 2 h at -20 ^oC. After fixation, cells were further treated with RNase A (10 mg/mL; Sigma-Aldrich) and stained with propidium iodide (PI; Sigma-Aldrich) for 30 min in dark at RT. The PI fluorescence of individual nuclei was measured using flow cytometer (RRID: SCR_002798; BD FACS Aria Fusion, BD BioSciences, SanJose, CA, USA).

Statistical analysis

All data were expressed as mean ± SD from three independent experiments and significance was checked by one-way analysis of variance (ANOVA) followed by Dunnett’s Multiple test using Graph Pad prism software 5.01 (GraphPad Software Inc., San Diego, CA, USA), p < 0.05.

Protein Profiles of Extact(s)

A high protein content of 3.8 mg/mL was found in CPMA as compared to CSMA and HFMA, containing 3.06 mg/mL protein. Furthermore, the protein separation of extract(s) using SDS-PAGE showed the presence of low molecular weight proteins ranging from 15.2 to 69.1 kDa in CPMA, 14.3 to 70.2 kDa in CSMA and 14 to 97.5 kDa in HFMA (Fig. 1) in which bands below 29.0 kDa were more prominent. The MW distribution of the proteins as well as its concentration was found to be higher in HFMA versus CPMA and CSMA.

Cytotoxicity of Skin Mucus Acetic Extract(s)

The cytotoxic effect of skin mucus acetic extract(s) was tested at different concentrations against A549 and NRK-52E cells. CPMA, CSMA and HFMA exhibited a significant dose dependent inhibitory effect on cellular proliferation of A549 cells at 400–800 µg/mL (Fig. 2a) with IC₅₀ values of 619, 591 and 538 µg/mL, respectively. HFMA was found to be the most effective against A549 cells as compared to CPMA and CSMA. Interestingly, all the three extracts exhibited no significant effect against the non-cancer NRK-52E cells up to 900 µg/mL (Fig. 2b)

Morphology of the treated and untreated cells

Treatment of A549 cells with 500, 600 and 700 µg/mL of CPMA, CSMA and 400, 500 and 700 µg/mL HFMA, revealed a dose dependent reduction in cell density, suggesting the anti-proliferative effect of the above extract(s). Hallmark characteristics of apoptosis viz. membrane blebbing, rounded and distorted cell morphology were also observed. Untreated A549 cells exhibited typical adherent cell morphology (Fig. 3). On the other hand, both control and treated NRK-52E cells exhibited normal epithelial cell adherent morphology up to 900 µg/mL of CPMA, CSMA and HFMA (Fig. 4).

Nuclear condensation/fragmentation

DAPI was used to observe nuclear apoptosis in A549 cells treated with 500, 600 and 700 µg/mL of CPMA/CSMA and 400, 500 and 700 µg/mL of HFMA. Figure 5.1 shows the photomicrographs of treated cells, displaying dose-dependent increase in the number of condensed or fragmented nuclei as indicated by the arrows. In contrast, no nuclear condensation was observed in untreated (control) A549 cells. Moreover, the quantitative measurement of apoptosis showed significant, dose dependent increase in percent apoptotic cells, as shown in Fig. 5.2. CSMA at 500, 600 and 700 µg/mL induced higher percentage of cells undergoing apoptosis viz. 25, 44.44 and 78.94%, respectively as compared to CPMA (20.28, 34.92 and 72.22%). Interestingly, HFMA treatment induced 27.7, 50 and 85.33% apoptotic cells at 400, 500 and 700 µg/mL, respectively indicating the highest apoptosis inducing potential of HFMA as compared to CPMA and CSMA.

Intracellular ROS generation

DCF-DA was used to compare intensities of ROS generation in A549 cells after treatment with 500, 600 and 700 µg/mL of CPMA/CSMA and 400, 500 and 700 µg/mL of HFMA. Photomicrographs revealed a dose-dependent enhancement of ROS generation in all extract(s) treated groups compared to untreated groups (controls) which showed little or no fluorescence (Fig. 6.1). Moreover, the quantitative measurement of ROS showed significant, dose dependent increase in percent fluorescence intensity (Fig. 6.2). Increased ROS generation viz.19.81%, 37.74% and 65.66% was observed in A549 cells treated with CSMA at 500, 600 and 700 µg/mL, respectively vis-à-vis 15.66%, 32.35% and 54.08% for A549 cells treated at same doses with CPMA. Interestingly, HFMA caused increase in fluorescence intensity by 47.56% and 69.81% at 500 and 700 µg/mL and was found to be the highest among the three extract(s). The results indicate that acetic extract(s) of fish skin mucus caused cellular damage to A549 cells through enhancement in ROS generation.

Induction of apoptosis

AO-EtBr dual staining of cells allows for distinction of viable, early or late apoptotic cells as apoptosis is accompanied by a series of morphological modifications. While AO is excluded by live cells, EtBr is taken up by live and dead cells alike. Figure 7a, b and c shows uniformly stained rounded green nuclei in the control live cells. At 500 µg/mL of CPMA/CSMA and 400 µg/mL of HFMA, there was a decrease in the number of green coloured viable cells, as the cells entered the early apoptotic phase indicated by greenish yellow or yellow coloured fragmented or condensed nuclei. More number of early apoptotic cells was observed in HFMA than CSMA and CPMA. At 600 µg/mL of CPMA/CSMA and 500 µg/mL of HFMA, more number of cells entered late apoptosis, as indicated by orange coloured fragmented nuclei. At 700 µg/mL of CPMA, CSMA and HFMA, all cells entered late apoptotic phase indicated by orange stained nuclei with a very few viable or early apoptotic cells. The results confirm that CPMA, CSMA and HFMA induced programmed cell death in A549 cells in a dose dependent manner, with HFMA having the highest cytotoxic effect.

Cell cycle arrest

Flow cytometry was used to analyse cellular DNA content of A549 cells in different phases of cell cycle when treated with different doses of the extract(s) (Fig. 8). All extracts caused an increase in SubG₀ population at higher doses compared to lower doses and untreated control, suggesting that there is an increase in apoptotic population. In the G₀/G₁ phase, only CSMA increased the percentage of cells at both the doses (68.5% at 700 µg/mL and 66.5% at 500 µg/mL) vis-à-vis control (64.7%), while other extracts (CPMA and HFMA) caused decrease in the proportion of cells compared to the control. CPMA at 500 µg/mL (14.6%) and HFMA at both concentrations (13.7% at 400 µg/mL and 13% at 600 µg/mL) caused significant accumulation of cells in S phase in comparison to the control (11.8%). In G₂/M phases, CSMA at 500 µg/mL (15.2%) and CPMA at 500 µg/mL (11.6%) caused the highest increase in percentage of cells as compared to the control (6.4%), followed by HFMA at both the doses (8.9% at 600 µg/mL and 7.3% at 400 µg/mL) and CSMA at 700 µg/mL (7.2%), while no significant change was observed at 500µg/mL (6.5%) of CPMA. The results suggest G₂/M phase arrest at all the doses of the three extract(s) except at 700 µg/mL of CPMA where no significant change was observed. Moreover G₀/G₁ arrest was also observed at both doses of CSMA. The cells which cannot be repaired due to high DNA damage undergone apoptosis at 400 and 600 µg/mL of HFMA and 700 µg/mL of CPMA and CSMA.

FSM is transforming into a topic of interest in biological research by implementing new avenues for drug development in the future, possessing an unexpected range of pharmacological properties. It is reported for killing and, preventing the growth and multiplication of human pathogens while the exploration of its anticancer potential is in its lag phase. The FSM of marine species Dasyatis pastinaca and Tachysurus dussumieri, inhibit the cell proliferation of HL-60 and A549 cells respectively [21, 49]. Lactose-binding molecules in the Korean eel skin mucus exhibit anticancer activity against K562 Cells [20]. Moreover, the FSM of freshwater species like Anabas testudineus, Misgurunus anguillicaudatus, Mughil cephalus, Neogobius melanostomus, Cyprinus carpio and the Indian major carps have also been reported to be cytotoxic to a variety of cancer cell lines [11, 12, 23, 50, 51].

The present study embarks on the pioneer exploration of cytotoxicity of CPMA, CSMA and HFMA on human A549 cells and their underlying mechanisms of cell death via apoptotic process. This study also investigated the protein profiles of the extract(s) which may have an active role in inducing the cytotoxicity. The result of protein quantification reveals the presence of significant amount of proteins in acetic extract(s) of the FSM. Proteinaceous nature of FSM of the selected fish species and various other freshwater species has also been reported by Wei et al. [13], Dhotre et al. [52], Elavarasi et al.[14]; Ramesh [53]; Rao et al. [54] and Bhatnagar et al. [37].

Previous studies have displayed the SDS-PAGE protein separation of FSM. Protein bands of 16 to 18 kDa have been reported in the eel skin mucus [20], 6.5 to 205 kDa in T. dussumieri skin mucus [49] and a wide range of distribution of proteins in Anabus testudineus skin mucus [12]. Likewise, the present study also demonstrated the presence of low MW protein bands in the acetic extract(s), which were more prominent below 29.0 kDa. The range of protein bands obtained in CPMA (14.3 to 70.2 kDa) is very similar to that reported by Loganathan et al. [55] in ammonium precipitate peptide mucus sample of C. striatus.

Furthermore, the anticancer assays showed that CPMA, CSMA and HFMA had no toxic effect against non-cancer NRK-52E cells while inducing a significant dose dependent cytotoxic effect against lung carcinoma A549 cells within the studied range. The MTT assay was performed and it was found that IC₅₀ value was lower for HFMA (538 µg/mL) compared to CSMA (591 µg/mL) and CPMA (619 µg/mL) against A549 cells. Likewise, in a previous study FSM of three freshwater species: Cirrhinus mrigala, Cyprinus carpio and Labeo rohita decreased the viability of A549 cells with an IC50 value of 735, 855 and 875 µg/mL respectively [11]. The FSM of freshwater species has also been reported to be cytotoxic to other cancer cell lines like LNCap (prostate cancer), MCF7 (breast cancer), A375.2S (melanoma) and HL-60 (leukemia) and non-toxic to normal fibroblast cells [12, 23, 50]. This specificity is may be due to the presence of proteins like lectins, buforinIIb and pleurocidin-like peptides which have reported to possess tumor-specific cytotoxic effect, which may be attributed to the negative charge of the cell membrane of cancer cells in contrast to zwitterionic property of cell membrane in non-cancer cells [56] and/or the disruption of the mitochondria [57].

The cellular morphology of A549 cells treated with the extract(s) showed shrinked and round shaped cells along with the detachment from surface which defines the characteristic features of onset of apoptosis [58]. Decrease in the cell number with increase in the dose of the extract(s) has been observed in which HFMA exhibited highest anti-proliferative effect, followed by CSMA, and CPMA was found to be least effective among the three extract(s). The occurrence of apoptosis is further investigated and the major apoptotic events were analysed under fluorescence microscope. A549 cells treated with the acetic extract(s) of FSM, on staining with DAPI dye displayed increasing levels of nuclear condensation/fragmentation with increase in the doses; the magnitude of condensation was highest in HFMA followed by CSMA and then CPMA. Interestingly, a similar dose dependent condensation of nucleus was reported in human leukemic K562 cells and human colon cancer HT 29 cells when treated with 50–500 µg/mL of Eel skin mucus [20] and 100–1000 µg/mL of T. dussumieri skin extract [59] respectively.

Moreover, the different stages of apoptosis were observed in A549 cells treated with the FSM acetic extract(s) at different doses. At 500µg/ml of CPMA and CSMA, early apoptotic features were observed, while both early and late apoptotic features were observed at the same dose of HFMA. At 400 µg/ml of HFMA, only early apoptotic cells were observed, similar to 500 µg/ml of CPMA and HFMA. Late apoptotic cells were observed in all the three extracts at 700 µg/ml. This indicates that, at lower doses HFMA is more effective than CPMA and CSMA in triggering the process of apoptosis. Similarly, Raja et al. [59] also observed early and late apoptotic cells in HT 29 cells treated with the skin extract of T. dussumieri. Apoptotic cell death is further confirmed by intracellular ROS generation in FSM acetic extract(s) treated A549 cells. ROS has been recognised as a crucial factor in triggering apoptosis in cancer cells, its overproduction damages the cell membrane, cytoskeleton, enzymes, proteins and DNA [60, 61, 62]. Various anticancer compounds derived from natural sources lead to apoptosis of cancer cells by inducing overproduction of ROS [63, 64]. Quantitative analysis of ROS generation by extract(s) followed the order HFMA > CSMA > CPMA indicating oxidative stress generation in cancer cells by the above extract(s).

DNA damage in the cells leads to activation of checkpoints which ultimately arrests the cell cycle. The cell cycle arrest prevent the cells containing damaged DNA to enter into the mitosis and provide sufficient time to the cells to either repair the damaged DNA or to undergo apoptosis if the damage is severe [65, 66]. In the present study, CPMA treated cells arrested the cell cycle in G₂/M phase in the low dose (500µg/mL), while directly undergoing apoptosis at a high dose (700 µg/mL). In CSMA treated cells, G₀/G₁ and G₂/M phase arrest was observed at low dose (500 µg/mL) while at high dose (700µg/mL) maximum numbers of cells are undergoing apoptosis with few cells being arrested at G₀/G₁ and G2/M phase. These results suggest that high dose of CPMA and CSMA, may be adequate to directly destroy the plasma membrane of cells while low doses may induce cellular stress that is leading to the cell cycle arrest at checkpoints rather than directly leading the cells to apoptosis. Moreover, in HFMA treated cells, both the lower and higher dose is causing cellular stress and cellular membrane disruption that is leading few cells to be arrested at G₂/M checkpoint of cell cycle while few cells are directly undergoing apoptosis. Our results are in agreement with Alijani et al. (2020), who also reported that low doses of Neogobius melanostomus and Cyprinus carpio skin mucus induced cellular stress in breast cancer cell line MCF-7 and prostate cancer cell line LNCaP respectively, while higher doses induced apoptosis.

The differential anticancer activities of the three skin mucus extract(s) might be attributed to difference in proportion of bioactive components present therein. It can be inferred from the results that cytotoxicity of the extracts is directly proportional to range of protein’s MW present in them; wider the range, higher is the anticancer effect. Therefore, HFMA possessing the widest range of proteins (14–97.5 kDa) is showing the highest anticancer activity compared to CSMA (14.3–70.2 kDa) and CPMA (15.2–69.1 kDa). The presence of low MW proteins in the extracts could be responsible for the anticancer activity of respective acetic extract(s). Future studies are required to identify and isolate the proteins and other bio-active compounds responsible for the observed anticancer effect, as well as testing the efficacy of fish skin mucus against various other types of cancer cell lines as well as in vivo models.

To the best of our knowledge, this is the first study conducted so far, evaluating the anticancer potential of skin mucus extracts of C.punctatus, C.striatus and H.fossilis. The acetic extracts of the mentioned fish species inhibited cell proliferation and induced apoptosis in A549 cells while no effect was observed on the growth of healthy NRK-52E cells. The findings of the present study provide a strong basis for further investigation and development of fish skin mucus as a promising therapeutic adjunct for cancer management along with the main line of treatment.

Funding

This work was supported by the UP-CST (Grant no. CST/375)

Conflict of interest

The authors do not have any pertinent conflicts of interest to declare.

Data availability

This manuscript does not report data generation or analysis

Acknowledgements

We thank Dr. Mohd Wamique from the Department of Medicine, King George’s Medical University, for his assistance in carrying out the SDS-PAGE and Central Drug Research Institute for providing facility of flow cytometry

Author Contributions

F.J. collected and analysed the data, and drafted the original manuscript. F.J. and P.A. designed and conducted the experiments, reviewed and edited the manuscript. R.A. and S.S. helped in data curation, critically reviewed and suggested modifications. M.S. provided the research concept, supervised the study and proofread the manuscript. All the authors discussed the results, gave feedback, and approved the final manuscript.

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Skin mucus extract derived from Channa punctatus, Channa striatus, and Heteropneustes fossilis induces apoptosis and suppresses proliferation in human adenocarcinoma cells via ROS mediated pathway and cell cycle arrest

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