Anticancer Potential of Synthesized Silver Nanoparticles Using Bioactive Metabolites of Actinobacteria Against A549 Lung Cancer Cells

Multi-drug resistance microorganisms and the rising numbers of cancer cases possess a critical threat to humankind, thereby motivating research on new weapons to combat the problem. To address these issues, researchers are now focusing on secondary metabolites produced by bacteria. Because of having outstanding antibiotic capabilities, actinobacteria are being explored as a potential solution to this problem. Silver nanoparticles of actinobacteria are green, eco-friendly, and cost-effective, as well as having antibacterial and anti-cancer properties and potential use in pharmaceuticals. Antibacterial potential of secondary metabolites produced by actinobacteria namely Microbacterium proteolyticum LA2(R) and Streptomyces rochei LA2(O) has been demonstrated by the well diffusion method. GC-MS was used to detect compounds in bioactive metabolites. The most abundant compound found in metabolites was n-hexadecanoic acid. UV-Vis spectroscopy was used to determine the extracellular development of silver nanoparticles of actinobacteria secondary metabolites, while transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) were used to inspect their morphological appearances, stability, crystalline structure, and coating. The activity of anticancer was assessed using the MTT test to evaluate the cytotoxicity of the secondar metabolites (pure and nanoparticles) against A549 lung cancer cells. We also evaluated the effect of secondar metabolites (pure and nanoparticles) on Reactive oxygen species (ROS) levels, Mitochondrial membrane potential (MMP) and Chromatin condensation through DCFDA, Mito Tracker and DAPI staining. Our result suggested that their secondary metabolites can be used a potential lead compound against cancer. However, more investigation is needed to totally grasp their mechanism of action. The MTT assay was used to determine cytotoxicity of compounds (LA2(O), LA2(R), LA2(ON), and LA2(RN)) against a cell line of human lung cancer A549. The secondary metabolites (LA2(O), LA2(R)), and their silver nanoparticles (LA2(ON), LA2(RN)) cytotoxic activity was compared to that of 5-FU. In this investigation, 5-FU, a notable anticancer medication, was employed as a positive control. It acts by preventing the synthesis of thymidine, resulting in cell death. Our results indicate that the compounds signicantly decreased A549 cell viability in under 24 hours. The results prove that the compounds. The IC 50 for 5-FU(a), LA2(O) (b), LA2(R) (c), LA2(ON) (d), and LA2(RN) (e) was obtained at 30 μM (52.73% cell viability), 125 µg (52.35% cell viability), 100 µg (52.3% cell viability), 30 μM (50.25% cell viability), and 70 µM (48.72% cell viability), respectively (Figure 6 a-e).


Introduction
Approximately 10 million deaths occur each year as a result of cancer, which is the subsequent biggest reason of mortality in the globe. According to the WHO report, the principal cause of cancer death in 2020 was Lung cancer (WHO, 2020). The resistance of chemotherapeutic drugs is arising in cancer cells, so lung cancer needs various therapy lines for treatment. Despite advances in elucidating the molecular Novel antibiotics and anticancer medicines with unique structures and properties have recently been discovered as the product of research on actinobacteria (Manivasagan et al. 2014; Ravikumar et al. 2012). While these cancer-ghting medications may have secondary effects such as requiring higher doses, and weaker blood ow for osteosarcoma patients, a patient's genetic pro le can in uence drug use outcomes. Recently, synthesized nanoparticles have emerged as a critical tool in cancer treatment. In addition to decreasing side effects, they have the ability to improve pharmaceutical delivery or absorption, or reduce toxicity in surrounding tissues. In addition to decreasing side effects, they have the ability to improve pharmaceutical delivery or absorption, or reduce toxicity in surrounding tissues. Some of the biological synthesis methods for nanoparticles provide considerable advantages, like being inexpensive, easy, and creating nanoparticles under a physiologically safe pH and room temperature.

Materials And Method
Isolation of actinobacteria A total of ve soil samples have been obtained from rhizosphere of medicinal plants i.e., Asparagus racemosus, Withania somnifera, Salvia o cinalis, Rouwol a serprntina, and Ocimum sanctum from different locations of Lucknow, Uttar Pradesh, and kept in sterilized plastic bags at 4ºC (Lee et al. 2014).
Eleven actinobacterial isolates representing different colony morphologies were isolated and cultivated on actinomycetes isolation agar for ve to seven days at ±28℃ (Sharma and Thakur 2020).
Preliminary Antimicrobial screening of Actinobacteria Primary screening was done by single streak of positive isolate of actinobacteria against pathogenic and Multi drug resistant (MDR) microbes streak at 90° on Mueller Hinton agar (Elbendary et al. 2018). The actinobacterial isolates were streaked as a parallel line on Mueller Hinton agar for bacteria and potato dextrose agar plates for fungi and incubated at 28˚C for 4-6 days (Ganesan et al. 2017). When actinobacterial strains properly grown on media, selected pathogenic bacterial strains i.e., Staphylococcus aureus (ATCC-6538), Pseudomonas aeruginosa (NCIM-5029), Salmonella abony (ATCC-6017), Klebsiella pneumoniae (NCIM-2957), Bacillus subtilis (MTCC-441) and Escherichia coli (MDR) (ATCC-25923) and pathogenic fungi i.e., Aspergillus niger (ITCC 545), Aspergillus avus (MTCC277), Aspergillus parasiticus (MTCC-2796) were streaked at right angles to the previous streak of actinobacteria and incubated at 30˚C. The A measurement of the zone of inhibition was taken after 72 hours for fungi and 24 hours for bacteria. Seven actinobacterial isolates were found positive after primary screening and subjected to molecular characterization.

Molecular characterization of positive isolates
The selected isolates from the secondary screening were subjected to molecular characterization using 16S rRNA sequence ampli cation performed at Bio-kart India Pvt. Ltd, Bangalore. The 16S ribosomal sequence ampli cation was conducted using the primers F243 (5'GGATGAGCCCGCGGCCTA3') and 1378R (5'CGGTGTGTACAAGGCCCGG 3'). Subsequently, the construction of phylogenetic tree by MEGA6 software, by applying the neighbor-joining DNA distance algorithm. The results of the microbial characterization revealed two isolates, i.e., LA2(R) Microbacterium Proteolyticum (MN560041) and LA2(O) Streptomycetes rochei (Zothanpuia 2015;Ganesan et al. 2017).

Production of secondary metabolites
The primarily screened actinobacteria with antimicrobial activities were used for the extraction of Green synthesis of silver nanoparticle Silver nanoparticles in-vitro production, was carried out by using the 1 mM aqueous solution of 50 μl AgNO 3 , that was pre-mixed with 50 ml supernatants of actinobacteria at 8.5 pH (Abd-Elnaby et al. 2016).
In rotary shaker at 200 rpm, and suspension was incubated in the dark at 37°C. for 5 days. To evaluate whether bacteria are involved in nanoparticle creation, the control tests involved running the process using un-inoculated medium and AgNO 3 solution. Silver ions reduction was tested by taking samples at speci ed intervals, using a UV-Vis spectrophotometer, and monitoring the UV-Vis spectrum. The color of silver nitrate solution changed to yellowish brown in each reaction vessel, which was then incubated with actinobacteria supernatant (

MTT assay
To check the feasibility of cells, an MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was accomplished. It is a colorimetric assay used to determine the activity of cellular enzymes responsible for reduction of the tetrazolium dye MTT to formazan crystals, which results in a purple color.

Statistical evaluation
One-way ANOVA was performed using Graph Pad prism 5 for statistical analysis on the data. The results were provided as the mean standard deviation of at least three independent measurements (nonsigni cant (ns), *p 0.05, ** p 0.01, *** p 0.001 against the untreated control).

Isolation of actinobacteria
Five rhizospheric soil samples of medicinal plants were air-dried and pretreated with CaCO 3 and then serial dilution was carried up to 10 -5 factors, spread on speci c agar and pure colonies were isolated by streak plate technique. After streaking, eleven pure isolates were found initially according to the morphological appearance of the colonies. The isolated isolates were lamentous, Gram-positive, nonmotile, and aerobic, with Catalase and Oxidase activity.

Preliminary screening and metabolite production
During preliminary screening, all 11 actinobacteria were tested for their ability to produce antimicrobials against pathogenic microorganisms. With the help of the perpendicular streak method, actinobacteria are tested for antimicrobial activity and seven strains are found positive.

Molecular characterization of positive isolates
All seven pure and active microbial strain cultures chosen during the primary screening experiments are further subjected for molecular characterization and found con rmation of two positive isolates named LA2(O) and LA2(R) using 16S rRNA sequence ampli cation. Further, the phylogenetic analysis con rmed that the isolates are Microbacterium proteolyticum LA2(R) and Streptomyces rochei LA2(O). The phylogenetic tree of the isolate Streptomyces rochei LA2(O) is characterized in Fig 1(a). The partial 16S rRNA gene sequence of isolate LA2(R) has been submitted in the NCBI GenBank with the accession number MN560041. Phylogenetic tree of the isolate LA2(R) is depicted in Fig. 1(b).

Production of secondary metabolites
After fermentation of ISP-2 broth, supernatant is extracted using ethyl acetate. After liquid-liquid extraction, the solvent phase is separated and crude extract was obtained after evaporation of solvent with the help of rotatory vacuum evaporator and mixed with methanol and stored for further use ( g. 2).
The metabolite production by submerged state fermentation techniques shows good results of antimicrobial activity against Staphylococcus aureus (ATCC-6538) and Klebsiella pneumoniae (NCIM-2957) as shown in Fig. 3.

Gas chromatography-mass spectrometry
The metabolite extract of LA2(R) and LA2(O) was subjected to GC-MS analysis. Identi cation of the compounds was based on the peak area, molecular weight and formula and similarity index. The GC-MS analysis of ethyl acetate extract of Streptomycetes rochei LA2(O) and Microbacterium proteolyticum LA2(R) are shown in Fig. 4 (a) and 4 (b), respectively. The compounds result shown by GC-MS analysis are presented in the Table 1. n-Hexadecanoic acid was the major compound present in both extracts with 95% and 92 % similarity index, respectively.

Synthesis of silver nanoparticle
The AgNPs were synthesized by incubating 1 mM AgNO3 with 333 µg/ml of secondary metabolites LA2(R) and LA2(O) at 40°C for 48 hours. It is proposed that Secondary metabolites, which together possess a synergistic ability to generate reducing potential capable of neutralizing AgNO3 (aq) into Ag The secondary metabolites and their respective silver nanoparticles cause oxidative stress on A549 lung cancer cells Treatment with the compounds caused considerable cell death in A549 cells, according to the ndings. The purpose of this study was to investigate whether the cytotoxicity of the compounds is due to the generation of reactive oxygen species. After treating A549 cells with the compounds, ROS levels were measured using DCFDA (2, 7-dichlorodihydro uorescein diacetate) labelling for 24h (Fig. 7a). Image J software was used to calculate DCFDA staining uorescence intensity in relation to ROS production (Fig.  7b). The results demonstrated that the compounds compared to untreated, produced higher levels of ROS cells.  (Fig 7a-b). This data recommended that A549 cells' inhibition of growth in response to chemicals could be attributable to oxidative stress.
The secondary metabolites and their respective silver nanoparticles Reduce MMP to induce apoptosis in A549 cells An increase in Reactive oxygen species has been referred to mitochondrial damage as well as a reduction in MMP (Cheng et al. 2019) therefore it has been checked that whether the compounds affected Mito Tracker Red staining of mitochondrial content in A549 cells (Fig. 8a). Using Image J software, the uorescence intensity of Mito Tracker Red staining related to MMP depletion was quanti ed (Fig. 8b) DAPI staining of secondary metabolites and their respective silver nanoparticles Using DAPI staining, we looked at another characteristic of apoptosis: chromatin condensation. The compounds were introduced to A549 cells for 24 hours and then examined to see if they had any effect on DNA integrity. In comparison to untreated cells, uorescence pictures of DAPI staining revealed chromatin condensation in treated cells (Fig. 9a). 5-FU caused 1.49-fold, LA2(O) caused 1.55-fold, LA2(R) caused 1.15-fold, LA2(ON) caused 1.34-fold, and LA2(RN) caused 1.74-fold increase in DAPI uorescence intensity compared to untreated A549 lung cancer cells (Fig. 9b). Together these results determine that the compounds promote apoptosis in A549 lung cancer cells.

Discussion
The present study showed that the actinobacteria Microbacterium sp. LA2(R) and Streptomycetes sp. Apoptosis suppression is primary reason of cancer proliferation, and it can be inhibited by a variety of oncogenic routes. The apoptotic route is suppressed in cancer through a number of methods, including anti-apoptotic protein over-expression and pro-apoptotic protein under-expression. Most of these alterations result in innate resistance to chemotherapy, which is the most prevalent anticancer treatment.
Finding new compounds that have anticancer potential by stimulating the apoptotic pathway is essential in developing new promising anticancer therapies (Pfeffer and Singh 2018). In this study, we analyzed the anticancer potential of the secondary metabolites derived from Microbacterium sp. and Streptomycetes sp. on A549 lung cancer cells. Further, silver nanoparticles of the metabolites were made, and their anticancer potential on A549 cells were also assessed. Our results demonstrated that the secondary metabolites (LA2(O), LA2(R)), and their silver nanoparticles (LA2(ON), LA2(RN)) caused cytotoxicity on A549 cells. After exposing A549 cells to the chemicals, an increase in ROS was detected. In cancer cells, ROS buildup plays a key function in apoptosis induction and cell cycle arrest. Numerous chemotherapeutic medications demonstrates their pharmacological properties by generating signi cant amounts of reactive oxygen species (ROS), which leads to mitochondrial membrane damage and death (Vandamme et al. 2012). Thus, the consequences of the compound on MMP in A549 cells were investigated.
MMP depletion was detected in A549 cells treated with the drugs. At low quantities, ROS is produced inside mitochondria (Santucci et al. 2019) and referred to as a favorable feedback system, entitled "ROSinduced ROS release"(RIRR) continues this interface (Zorov et al. 2000). When mitochondria are subjected to deleterious events whereas suffering a decrease in transmembrane potential, uncontrolled ROS develops. RIRR causes mitochondrial ROS to be produced, which reduces MMP and causes mitochondrial permeability transition pores to open more slowly (mPTP) (Zorov et al. 2000). Mitochondrial membrane depolarization disrupts mitochondrial respiratory chain activity, this may result in the buildup of reactive oxygen species (ROS) . Apart from a drop in MMP, another distinct hallmark of apoptosis is membrane blebbing, chromatin condensation, and DNA fragmentation (Chang et al. 1997). Thus, we performed DAPI staining, and upregulation in DNA condensation was observed after treatment with the compounds in A549 lung cancer cells. Overall, our results demonstrated that the compounds were exhibiting early hallmarks of apoptosis. However, further studies are required to thoroughly understand their anticancer potential that may help identify new disease targets, resulting in the development of new treatment techniques for a wide range of disorders. Thus, our ndings suggest the anticancer potential of secondary metabolites of actinobacteria and their biosynthesized silver nanoparticles against A549 lung cancer cells that could play an important role in the development of new therapeutic agent for the treatment of cancer.
In short, the current study has showed that these novel and rare actinobacteria were able to produce a wide range of bioactive compounds which could serve as potential sources for future drug development. The present work provides helpful insight into the development of new antimicrobial agents with the synergistic enhancement of the antibacterial mechanism against pathogenic micro-organisms.

Statistical analysis
Statistics were presented as the average of three separate trials' standard deviations. The data analysis was carried out by using Origin 6.0 software, which was developed in the United States, as reported previously (Alvi et al. 2017).

Future perspectives
Green nanoparticle production seems to be in a research phase for cancer treatment and diagnosis, but drug trials are needed to be determined in its nal dependability. Due to their biocompatibility and effectiveness, green nanomaterials have opened up plenty of new options in terms of their application. Furthermore, numerous cancers that currently have no solutions may be treated in the future by these green nanomaterials. Given the foregoing, green nanomaterials are predicted to emerge as future cancer treatments and diagnostics compounds in the upcoming future.  The procedure of secondary metabolite production and its antimicrobial activity       Consequence of the compounds on chromatin stability in lung cancer cells was explored by DAPI staining. (a) Fluorescence micrographs depict chromatin condensation after treatment with the compounds for 24h in A549 lung cancer cells. (b) Image J software was used to quantify the relative change in uorescence intensity of DAPI staining in A549 cells on a graph. Scale . Key-5-FU: 5-Fluorouracil.