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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 200-208

Novel strain of Nocardiopsis sp. CN2 from andaman nicobar islands: Isolation, taxonomy, fermentation, and antiproliferative effect on cervical cancer cells

1 Centre for Advanced Study in Marine Biology, Annamalai University, Chidambaram, Tamil Nadu, India
2 Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology, Chennai, Tamil Nadu, India
3 School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
4 Department of Biochemistry and Biotechnology, Annamalai University, Chidambaram, Tamil Nadu, India
5 Department of Microbiology, Periyar University, Salem, Tamil Nadu, India

Date of Submission03-Apr-2020
Date of Acceptance02-May-2020
Date of Web Publication12-Sep-2020

Correspondence Address:
Dr. M Radhakrishnan
Centre for Drug Discovery and Development, Sathyabama Institute of Science and Technology, Chennai - 600 119, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_51_20

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Background: Cancer continues to be one of the world's deadliest diseases. New antibiotics with unique modes of action are urgently needed and therefore extensive research has been undertaken on novel anti-cancer antibiotics from natural resources. The aim of this present study was undertaken to characterize the marine actinobacterial strain CN2 isolated from Andaman and Nicobar Islands and also to evaluate its antiproliferative activity on HeLa cervical cancer cell line. Methods: Antiproliferative effect of the ethyl acetate extract was evaluated on HeLa cell lines by MTT assay and the oxidative stress was determined by lipid peroxidation and glutathione reductase changes in the antioxidant status. To understand the mode of antiproliferative effect, intracellular ROS levels by DCFH-DA method, mitochondrial membrane potential alterations by Rh-123 staining, oxidative DNA damage by comet assay, and apoptotic morphological changes by AO/EtBr-staining method were studied. Results: Based on the studied phenotypic, chemotaxonomic and molecular characteristics, strain CN2 was identified as Nocardiopsis sp. and it showed 99.9% similarity with N. exhalans (EU570349). However differences observed in certain phenotypic properties between strain CN2 and its close related species N. exhalans evidenced its novelty at strain level. Actinobacterial extract enhanced the ROS level, as evidenced by the increased DCF fluorescence. Further, it altered the mitochondrial membrane potential and increased the oxidative DNA damage and apoptotic morphological changes in HeLa cell lines. Conclusion: This is the first report on antiproliferative activity of the rare actinobacterium, Nocardiopsis sp. from Andaman and Nicobar Islands. Further production, purification and characterization of active metabolite from the ethyl acetate extract will pave the way for promising anticancer drug development.

Keywords: Cervical cancer, fermentation, marine actinobacteria, MTT assay, Nocardiopsis

How to cite this article:
Chandrakumar D, Manigundan K, Gopalakrishnan K, Karuppiah V, Prasad N R, Radhakrishnan M, Sivakumar K, Balagurunathan R. Novel strain of Nocardiopsis sp. CN2 from andaman nicobar islands: Isolation, taxonomy, fermentation, and antiproliferative effect on cervical cancer cells. Biomed Biotechnol Res J 2020;4:200-8

How to cite this URL:
Chandrakumar D, Manigundan K, Gopalakrishnan K, Karuppiah V, Prasad N R, Radhakrishnan M, Sivakumar K, Balagurunathan R. Novel strain of Nocardiopsis sp. CN2 from andaman nicobar islands: Isolation, taxonomy, fermentation, and antiproliferative effect on cervical cancer cells. Biomed Biotechnol Res J [serial online] 2020 [cited 2022 Jan 29];4:200-8. Available from: https://www.bmbtrj.org/text.asp?2020/4/3/200/294858

  Introduction Top

Cancer remains one of the highest causes of death globally. Cancer of the cervix uteri is the fourth most common cancer among women worldwide, with an estimated 527,624 new cases and 265,672 deaths in 2012[1]. The majority of cases are squamous cell carcinoma followed by adenocarcinomas. About 122,844 new cervical cancer cases are diagnosed and 67,477 cervical cancer deaths occur annually in India. Cervical cancer ranks as the second leading cause of female cancer as well as female cancer deaths in India. Treatment for cervical cancer includes surgery, chemotherapy, and radiation therapy. A great number of anticancer compounds which are currently available in the market are derived from microbial natural products or their derivatives. The global scientific community is investigating various natural resources including marine organisms to find new therapeutic leads for cancer treatment.

Actinobacteria are the economically and biotechnologically most valuable prokaryotes with the unparalleled ability to produce novel bioactive metabolites.[2] Actinobacteria are provided us with different classes of antibiotics that play a critical role in treating infectious and on infectious diseases.[3] Toward the aim of discovering new molecules displaying different biological activities, researchers around the world have dedicated time and efforts to looking for novel sources such as animal, microbes, and plants from varied environments including thermal springs, Antarctica, caves, and marine, to mention a few, from which to draw antibiotic leads. For several decades, the marine environments have drawing been attention, as many new compounds showing potential antibacterial activities have been discovered from bacteria.[4] In particular, actinobacteria from marine ecosystems are the producers of a large number of novel molecules with different biological activities, including antitumor properties. These antitumor compounds belong to several structural classes such as anthracyclines, enediynes, indolocarbazoles, isoprenoides, macrolides, nonribosomal peptides, and others. In this study, we characterized the actinobacterial strain CN2 and reported its antiproliferative activity against cervical cancer cell line.

  Materials and Methods Top

Description of actinobacterial strain CN2

The marine actinobacterial strain CN2 used in this study was isolated from the sediments collected from the tsunami degraded mangrove area of the Kimios Bay region, Car Nicobar, Andaman and Nicobar Islands (Lat. 9.1o N; Long. 92.7o E) using Kuster's agar medium. Viability of strain CN2 was maintained in yeast extract, malt extract dextrose agar slants, as well as in 30% glycerol broth.

Taxonomy of actinobacterial strain CN2

Phenotypic characteristics, namely cultural, micromorphological, carbon, and nitrogen source utilization characteristics of the strain CN2 were carried out by adopting standard methods.[5],[6],[7] Cell wall amino acids and whole sugars of strain CN2 were studied using the methods described by Lechevalier and Lechevalier (1970).[8]

Molecular characterization of strain CN2 was also done by 16s rDNA sequencing and phylogenetic analysis to determine its taxonomic position. Genomic DNA from the fresh culture actinobacterial strain CN2 grown in ISP2 broth was isolated by adopting the method described by Hopwood et al. (1985).[9] Polymerase chain reaction (PCR) was performed to amplify the 16sRRNA gene of strain CN2 using the universal primers 27F and 1492R. For the specific amplification of 16S rRNA fragments of actinobacteria, the reaction mixture contains 2 μl of the template DNA, 2 μl of forward primer 27F, 2 μl of reverse primer 1492R, 0.5 μl of dNTPs mix, 5 μl of Taq buffer with MgCl2, 1 μl of 5U Taq DNA polymerase, and 37.5 μl of molecular grade water. Amplification was done with the initial denaturation at 94°C for 8 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 53°C for 45 s, and annealing extension at 72°C for 2 min, with the final extension at 72°C for 10 min, and finally, the reaction was held at 4°C. Amplified products were analyzed by resolving the 10 μl of the PCR product with the loading dye in one lane and marker in another lane of 1.5% agarose gel.

The purified DNA sample was directly sequenced using an AmpliTag FS DNA sequencing Kit (Applied Biosystem). A sequence of 1463 bp was perforated. The data were analyzed using applied biosystem DNA editing and assembly software and sequence comparisons were obtained using the MicroSEQ Software.

Sequence similarity search was done for the 16S rDNA sequence of the strain CN2 by applying their sequence to BLAST search of the National Center for Biotechnological Information (NCBI), USA. Phylogenetic analysis was performed using the software package Molecular Evolutionary Genetics Analysis (MEGA) version 4[10] after multiple alignments of data by CLUSTAL_X.[11] A phylogenetic tree was reconstructed using the neighbor-joining method of Saitou and Nei (1987)[12] from Knuc values.[13],[14] The topology of the phylogenetic tree was evaluated using the bootstrap resampling method of Felsenstein (1985)[15] with 1000 replicates. The taxonomic position of actinobacterial strain CN2 was determined based on their phenotypic, cell wall and molecular characteristics.

Bioactive metabolite production from strain CN2

Strain CN2 was grown on yeast extract malt extract dextrose broth at 30°C in a shaker with 95 rpm. On the 5th day, the culture broth was centrifuged at 10,000 rpm for 15 min at 4°C. The secreted bioactive metabolite from the supernatant was extracted by liquid–liquid extraction method using an equal volume of ethyl acetate for 24 h at room temperature. Then, the solvent portion was collected and concentrated using a rotary evaporator. After evaporation, the concentrated crude extract was dissolved in 2 ml of dimethylsulfoxide (DMSO) and filter sterilized using Millipore filter (20 μm). This filter-sterilized extract was used as a crude sample for the antiproliferative assay.[16]

Determination of antiproliferative activity on cervical cancer cells

Cell lines

The HeLa cell line (human cervical cell line) was obtained from NCCS, Pune. HeLa cells were grown to 80% confluence in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum (FCS). The cells were subcultured and 105 cells were seeded on a fresh 6-well plate. After the monolayer of cells became confluent in the 6-well plates, cancer cells were treated with different concentrations of ethyl acetate extracts (10, 20, 30, 40, and 50 μg/ml) in DMEM without serum for 24 h. After the 24 h treatment, various molecular endpoints were evaluated in control and actinobacterial extract-treated cells.

MTT assay

After treatment with the ethyl acetate extract, the antiproliferative effect was evaluated using the MTT assay based on the cleavage of the yellow tetrazolium salt MTT to formazan crystals by the metabolically active cells.[17] The formazan crystals were solubilized by the addition of DMSO. Absorbance was measured at 570 nm. The treated as well as control HeLa cells were subjected to the following analysis.

Determination of apoptotic morphological changes

Staining of DNA with acridine orange (AO) and ethidium bromide (EtBr) allowed visualization of the condensed chromatin of dead apoptotic cells.[18] Stained cells were viewed under a fluorescence microscope. During apoptosis, DNA becomes condensed and fragmented. The number of cells showing features of apoptosis was counted as a function of the total number of cells present in the field.

Measurement of intracellular reactive oxygen species in cells

Both the treated as well as control HeLa cells were added with the fluorescent dye 2'-7'dichlorofluorescin diacetate (DCFH-DA) and kept in an incubator for 30 min. Then, the cells were washed with Phosphate-buffered saline (PBS) to remove the excess dye. Intracellular reactive oxygen species (ROS) was measured by using a nonfluorescent probe (DCFH-DA) that can penetrate into the intracellular matrix of cells where it is oxidized by ROS to fluorescent dichlorofluorescein (DCF).[19] Fluorescent measurements were made with excitation and emission filters were set at 485 ± 10 and 530 ± 12.5 nm, respectively. Fluorescence microscopic images were taken using a blue filter at 450–490 nm.

Alterations in mitochondrial membrane potential

After incubation of the cells with extract for 24 h, fluorescent dye Rh-123, a lipophilic cationic dye highly specific for mitochondria) (10 μg/ml), was added to the cells which were then kept in an incubator for 30 min.[20] Then, the cells were washed with PBS and viewed under a fluorescence microscope using a blue filter. Polarized mitochondria were marked by orange–red fluorescence and depolarized mitochondria, by green fluorescence.

Analysis of DNA damage

DNA damage was estimated by alkaline single cell gel electrophoresis (comet assay) according to the method of Chen et al. (2005).[21] For the analysis of the comet images, the extent of DNA damage was estimated by fluorescence microscopy using a digital camera and analyzed by image analysis software, CASP.[22] For each sample, 100 cells were analyzed and classified visually into one of the five classes according to the intensity of fluorescence (DNA) in the comet tail. DNA damage was quantified by the tail moment, tail length, and olive tail moment.[23]

Estimation of antioxidant enzyme activity

The supernatant obtained after centrifuging the trypsinized cells was used for the measurement of activities of antioxidant enzymes. The level of lipid peroxidation was determined by analyzing Thiobarbituric acid reactive substances (TBARS).[24] The levels of reduced glutathione (GSH) were determined in the supernatant according to the procedures described elsewhere.[25]

Statistical analysis

Statistical analysis was performed by one-way ANOVA followed by Duncan's multiple range test (DMRT) taking P ≤ 0.05 to test the significant differences between the groups.

  Results Top

Characterization and taxonomy of strain CN2

Actinobacterial strain CN2 formed an extensively branched substrate mycelium and aerial hyphae that differentiated into long spore chains [Figure 1]. Reverse side pigment and diffusible pigments were not produced on ISP2 agar. Melanin pigment was also absent on ISP7 agar. The utilization of the carbon and nitrogen sources showed that arabinose, inositol, mannitol, rhamnose, sucrose, raffinose, glucose, galactose, maltose, L-alanine, and gelatin were utilized, whereas xylose, fructose, acetate, paraffin, and serine were not utilized [Table 1].
Figure 1: Cultural and morphological features of potent strain CN2 (a: Whitish aerial mycelium; b: Long chain at × 400; c: Smooth surface in Scanning Electron Microscopy (SEM) image at × 15000)

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Table 1: Cultural and morphological characteristics of the strain CN2

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Chemical characters are being used increasingly in the classification and identification of bacteria and they have been responsible for some of the improvements in the taxonomy of the actinobacteria.[8] These organisms have been arranged into several groups on the basis of the limited distribution of certain aminoacids and sugars found in significant amounts in the peptidoglycan layer. The examination of whole-cell hydrolysates gives sufficient data for accurate identification. In the present study, strain CN2 showed the presence of meso-DAP in the peptidoglycan layer with no diagnostic sugar pattern, indicating that this strain belongs to the cell wall chemo Type III. The genera belonging to the wall Type III are Actinomadura, Microbispora, Streptosporangium, Spirillospora, Planomonospora, Dermatophilus, and Nocardiopsis.[8] The cultural and morphological characters and their carbon and nitrogen source utilization were also analyzed to identify the strain CN2.

Molecular taxonomy

DNA was isolated from the strain CN2, and 16S rDNA was amplified through PCR which showed the molecular weight of 1.5 kb using the universal primer 27F and 1492R. The amplified product was purified and it was sequenced. A 1481 bp of 16S rDNA sequence was determined for the strain CN2, which was submitted to the GenBank (National Center for Biotechnology Information, USA), and an accession number (HQ013308) was obtained.

Comparison of the 16S rRNA gene sequence (1481 bp) of the strain CN2 with previously obtained sequences of Nocardiopsis species deposited in GenBank (NCBI) indicated that this organism is phylogenetically related to the members of the genus Nocardiopsis.

A phylogenetic tree based on 16S rRNA gene sequences of members of the genus Nocardiopsis was constructed according to the neighbor-joining method with CLUSTAL W (version 1.81) and MEGA (version 7). For the neighbor-joining analysis, a distance matrix was calculated according to the Kimura's two-parameter correction model. The rooted phylogenetic tree indicated that the strain CN2 formed a distinct branch with the proposed type strains of Nocardiopsis metallicus, Nocardiopsis alba, and Nocardiopsis exhalans [Figure 2]. Strain CN2 exhibited 99.5% similarity to N. alba (X97884) 99.8%, similarity to N. metallicus (AJ420769) 99.9%, and similarity to N. exhalans (EU570349) [Table 2].
Figure 2: Phylogenetic tree based on 16S ribosomal RNA gene sequences showing the positions of CN2 and related strains. Only bootstra P values expressed as percentages of 1000 replications are shown at the branch points

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Table 2: Levels of 16S rDNA sequence similarity between the strain CN2 and the reference strains

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Phylogenetic position of this strain formed a distinct clade with the closest similarity with N. exhalans, N. alba, and N. metallicus. However, the strain CN2 differed considerably in biochemical characters with the closest phylogenetic members [Table 3]. Further, the analysis of DNA–DNA re-association, menaquinone, and phospholipids for the strain CN2 will correctly identify the species.
Table 3: Cultural, morphological, and physiological characteristics of strain CN2 and other member of the closely related Nocardiopsis species

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Antiproliferative activity of Nocardiopsis strain CN2

To investigate the growth inhibitory effect of CN2 extract, cell proliferation (MTT) assay was performed, and 10, 20, 30, 40, and 50 μg/ml of CN2 extract treatment significantly inhibited HeLa cells; 50 μg/ml of CN2 treatment showed only 15% cell viability [Figure 3]. Hence, for the further experiment, 10, 20, 30, 40, and 50 μg/ml of CN2 extract was chosen.
Figure 3: Effect of inhibitory effect of CN2 extract on HeLa cell line (MTT assay)

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The ROS level was measured using a nonfluorescent probe (DCFH-DA) that can penetrate into the intracellular matrix of cells where it is oxidized by ROS to fluorescent DCF [Figure 4]. The nonfluorescent DCFH-DA is oxidized by intracellular ROS and forms the highly fluorescent DCF which is measured spectroflurometrically at emission filters set at 485 ± 10 nm and 530 ± 12.5 nm, respectively. In the present study, there was a significant increase in the ROS levels up to 70% in 50 μg/ml of CN2 extract treatment in HeLa cells.
Figure 4: Fluorescence microscopic images of intracellular reactive oxygen species measurement by 2, 7,-diacetyl dichlorofluorescein (staining (a-f), Fluorescence microscopic images of mitochondrial membrane potential by Rh-123 staining (g-l), Fluorescence microscopic images of oxidative DNA damage (comet assay) (m-r), Fluorescence microscopic images of apoptotic morphology by dual staining (s-x)

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The alteration in mitochondrial membrane potential (ψm) is an indication of early stages of apoptosis. Rhodamine 123 (Rh 123) is a lipophilic cationic dye, highly specific for mitochondria. Polarized mitochondria are marked by orange–red fluorescence and depolarized mitochondria are marked by green fluorescence. Cont–rol cells appeared orange–red, whereas the CN2 extract-treated cells appeared green [Figure 4]. Rhodamine 123 is a lipophilic cationic dye that enters only live cells and stains mitochondrial DNA, and hence, the live cell mitochondria appeared orange–red under blue emission. In the results, CN2 extract-treated HeLa cells showed a significant decrease in mitochondrial membrane potential in cells exposed to 24 h incubation.

The single cell gel electrophoresis assay (also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell [Figure 4]. It has gained popularity as a standard technique for the evaluation of DNA damage/repair, biomonitoring, and genotoxicity testing. It involves the encapsulation of cells in a low-melting point agarose suspension, lysis of the cells in neutral or alkaline (pH > 13) conditions, and electrophoresis of the suspended lysed cells. This is followed by visual analysis with the staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software.

Fluorescent DNA-binding dyes including AO and EtBr were used to differentiate cells that are in stages of apoptosis and necrosis. Early-stage apoptotic cells take up AO but not EtBr. They are stained green, whereas nonviable cells take up both dyes and are stained orange. Since healthy cells also take up AO, the cells undergoing apoptosis are identified by analysis of their chromosome condensation or by observation of fragmentation that did not occur in healthy cells. AO/EtBr staining of lymphocytes treated with CN2 extract treatment showed condensed nuclei, membrane blebbing, and apoptotic bodies [Figure 5].
Figure 5: Percentage of apoptosis. The values are given as mean ± standard deviation of six experiments in each group. Bars not sharing the common superscripts differ significantly at P≤ 0.05

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The extent of DNA damage was calculated by % tail DNA, tail length, tail moment, and Olive tail moment in normal and CN2 extract-treated HeLa cells. CN2 extract-treated cells significantly increased % tail DNA, tail length, tail moment, and Olive tail moment in cultured HeLa cells [Figure 6].
Figure 6: CN2 extract induced DNA damage in HeLa cells. Comet parameters (% tail DNA [a], % tail length [b], tail moment [c], and olive tail moment [d]). The values are given as mean ± standard deviation of six experiments in each group. Bars not sharing the common superscripts differ significantly at P≤ 0.05

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Lipid peroxidation has been used as an endpoint to study the action of prooxidant as well as to investigate the effect of antioxidants. The study of lipid peroxidation is attracting much attention in recent years due to its role in disease processes and oxidative stress. In this study, levels of a thiobarbituric acid reactive substance (TBARS) were increased significantly in >50 μg/ml of CN2 extract-treated HeLa cells. CN2 extract treatment showed progressively increased levels of TBARS in a concentration-dependent manner.

GSH is the important intracellular reducing species in cells that detoxify hydrogen peroxide. It plays an important role in the antioxidant defense and it scavenges free radicals produced by cytotoxic drugs. GSH plays an important role in modulating the effect of chemotherapy and radiotherapy, and the alterations in its concentration can drastically affect the response of the tumor. The depletion of intracellular thiols is the obvious approach to sensitize cells to the cytotoxic effect of CN2 extract [Figure 7]. A much more sophisticated and controllable approach to depleting intracellular thiols is the inhibition of steps in their biosynthesis.
Figure 7: Effect of CN2 extract on GSH level. The values are given as mean ± standard deviation of six experiments in each group. Bars not sharing the common superscripts differ significantly at P≤ 0.05

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  Discussion Top

The main difficulty in the battle against cancer is the limited specificity of current chemical treatments; thus, to address this problem, increasingly more researchers are seeking to isolate and screen natural compounds that can effectively target cancer cells, without causing undesirable adverse side effects on normal cells. A number of anticancer agents have been isolated as natural compounds from plants, animals, and microorganisms.[26],[27] Examples of such agents currently used in the treatment of different forms of cancers are vincristine, vinblastine, taxol, actinomycin, and bleomycin. With nearly two million recognized living species that exhibit enormous chemical diversity, nature offers a potentially huge source of new therapeutic compounds. For many organisms, at least a fraction of this chemical diversity seems to be the result of evolutionary selection associated with defense and/or predatory mechanisms. In this regard, actinobacteria play a major role in producing secondary metabolites and marine actinobacteria have furnished new and unique active principles.[28]

In this study, anticancer effect of CN2 extract was studied against the cervical cancer cell line in vitro. CN2 extract was cytotoxic in a dose-dependent manner in HeLa cell line. Maximum cytoxicity of the CN2 extract was observed at 24 h incubation, and it worked in a concentration-dependent manner. The mechanism of action of CN2 extract on the tumor cell cytotoxicity was not necrosis based but involved apoptosis or apoptosis-like programmed cell death (PCD). Possibility to induce apoptosis by physical and chemical inducers in HeLa cells has been well-documented.[29] Distinctive events of apoptosis are appreciable cytoplasmic shrinkage, condensation of chromatin, cleavage of DNA into 180–200 bp nucleosomal units, activation of proteolytic enzymes known as caspases, and disintegration of the cells into small fragments. The present study indicated that the mechanism of action of CN2 extract on HeLa cells fully conforms to this apoptotic pattern with regard to cytoplasmic and nuclear changes. HeLa cells did not undergo apoptosis under the experimental conditions used in this work (untreated control), but clear apoptotic morphological changes were observed in CN2 extract-treated cells. This clearly indicates the apoptotic inducing property of CN2 in HeLa cells.

It appears that the biochemical injuries make cancer cells more vulnerable than normal ones to pro-apoptotic events such as mitochondrial membrane permeabilization and release of apoptosis-inducing factors, and ultimately, more exposed to the action of pharmacological agents.[30] In this study, decreased mitochondrial membrane potential was observed in CN2-treated cells. Others reports stated that it is likely that drugs targeting the respiratory chain components might be more toxic to tumor cells.[31] Among the effects derived from the inhibition of the mitochondrial respiratory chain complex I, an effective blockage of cell proliferation and an increase in the generation of ROS leading to apoptosis induction are important.[32] In the same line, increased ROS levels in CN2 extract-treated cancer cells were observed.

The results of the present study indicate that CN2 extract induced apoptosis is preceded by intracellular ROS generation. This increased ROS generation may induce oxidative DNA damage in CN2 extract-treated cells. Significant DNA damage (% tail DNA, tail length, tail moment, and Olive tail moment) was observed in CN2 extract-treated HeLa cells. DNA is an important target in cancer cell killing. The induction of double-strand breaks and single-strand breaks is often predicted for the cancer cell death. There were increased lipid peroxidation (TBARS) and decreased GSH levels in CN2 extract-treated cells. This increased TBARS and decreased GSH levels in HeLa cells may also be associated with ROS inducing property of CN2 extract.[33]

In the present study, the prooxidant mechanism of CN2 extract in HeLa cells has been clearly indicated. Altogether, the results suggest that in HeLa cells, viability decrease and cellular apoptosis induced by CN2 extract are mediated by ROS, and at least the mitochondrial pathway may be triggered by the CN2 extract.

Experimental evidences show that a reduction in mitochondrial membrane potential constitutes an irreversible step of PCD.[34] In fact, it is well known that the loss of mitochondrial membrane potential can induce the opening of permeability transition pores in the mitochondria and the release of small molecules from the intermembrane space that acts as cell death-promoting factors, promoting caspase-dependent and caspase-independent apoptosis. Among these factors, cytochrome c (cyt-c) and apoptosis-inducing factor (AIF) play a pivotal role in determining PCD.[34] Cyt-c induces caspase-dependent apoptosis by triggering the assembly of apoptosome from apoptotic protease-activating factor 1, ATP, and procaspase-9, which can then activate effector caspase-3. On the other hand, AIF is the main mediator of caspase-independent apoptosis-like PCD, and after translocation to the nucleus, it induces chromatin condensation and large-scale DNA fragmentation.[33] In this study, chromatin condensation and large-scale DNA fragmentation have been observed in CN2 extract-treated HeLa cells. Loss of mitochondrial membrane potential, chromatin condensation, and DNA damage are compatible with the hypothesis that AIF may be involved in apoptosis-like PCD induced in HeLa cells by CN2.

Taken as a whole, experimental data reported in the present study suggest that in HeLa cells, the CN2 extract exerts its action by triggering a mitochondrion-dependent apoptotic program. In the opinion of some researchers, use of drugs that target mitochondria in tumor cells to initiate mitochondrial-activated apoptosis could be a pivotal strategy for cancer chemoprevention or therapy.[35] In line with this, the results obtained in the present study may represent the point of departure for more extensivein vitro investigations and furtherin vivo research to fully understand the mechanism of action of CN2 extract and to evaluate its potential use in cancer chemotherapy.

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Conflicts of interest

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  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]

  [Table 1], [Table 2], [Table 3]


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