DNA damage induced cellular senescence and it’s PTEN-armed exosomes—the warriors against prostate carcinoma cells

Exosomes are one type of small extracellular vesicles (EVs) having a size range of 30–150 nm and secreted by the endosomal compartment of most eukaryotic cells. It has been found that exosomes (EVs) can serve as a communicating vehicle to transfer information among cells and thus can be associated with numerous physiological and pathological functions. In this study, we have isolated exosomes (EVs) from two different human cancer cell lines. Isolated exosomes (EVs) were characterized by scanning electron microscopy, nanoparticle tracking analysis, DLS, and by western blotting. It was observed that exosomes (EVs) isolated from mock-treated human lung epithelial carcinoma (A549) cells or HeLa cells exerted growth arrest to the human prostate carcinoma (PC3) cells, but no growth arrest was observed in case of normal human lung fibroblast cell line (WI-38). Additionally, exosomes (EVs) isolated from PC3 cells have no effect on PC3 cells. This is also true for exosomes (EVs) isolated from H2O2-induced senescent human lung cancer cells (A549). Analysis of exosome (EVs) content by western blotting reveals the presence of PTEN in the exosome (EVs) of lung cancer cells. Functional analysis of PTEN pathways in PC3 cells indicates the inactivation of Akt in exosome (EV)-treated cells. Therefore, from our study we have concluded that exosomes (EVs) secreted from A549 cells which contain functional PTEN may be used for delivery of PTEN to cancer cells without functional PTEN.


Introduction
Exosomes are small-sized extracellular vesicles (EVs) surrounded by phospholipid bilayer and their size ranges are approximately 30-150 nm [1,2]. Almost all kinds of cells are capable of secreting exosomes. While being released from the cells, exosomes carry some components, such as proteins, mRNA, microRNA, siRNA, and DNA [3]. It has been found that exosomes are less immunogenic and noncytotoxic to the recipient cells [4]. This makes them a potential carrier of drugs and other therapeutic agents to treat different types of diseases in recipient cells. Exosomes after fusion with recipient cells release the cargos into that cell.
Thus, they play an important role in cell to cell communication [2]. The most important cargoes that exosomes carry are the proteins which can provide crucial information correlated with the physiological states of the cells from which exosomes originate. Proteins that are found in exosomes include membrane transport and fusion proteins, such as annexins, flotillins, GTPases, heat shock proteins, tetraspanins, proteins involved in multivesicular body biogenesis, lactadherin, platelet derived growth factor receptor, transmembrane proteins, and lysosome-associated membrane protein-2B, as well as lipid-related proteins and phospholipases. These proteins can therefore be used as biomarkers for isolation and quantification of exosomes [1].

Cell culture and treatment
PC3 (Human prostate carcinoma cells), A549 (Human lung carcinoma cells), HeLa (Cervical carcinoma cells), and WI-38 (Normal lung fibroblast cells) were purchased from the National Center for Cell Science, (Pune, India) and maintained at 37 °C, 5% CO 2 , and 95% relative humidity (RH) in RPMI 1640 and DMEM media, respectively, supplemented with 10% FBS and/or 10% Exosome-depleted FBS, penicillin-streptomycin (100 units/ml), and amphotericin-B (anti-fungal). Cells were seeded overnight in two 60-mm culture plates in media supplemented with 10% FBS. Then, media of both the plates were withdrawn and fresh media supplemented with 10% exosome-depleted FBS were added to the plates. One of the plates were treated with hydrogen peroxide (H 2 O 2 ) (150 µM for 48 h) and the other was kept as untreated.

Isolation of exosomes (EVs)
To isolate EVs from cell culture supernatants, cells were cultured in medium containing 10% exosome-depleted FBS. Culture supernatants were centrifuged at 300 × g for 10 min at room temperature to remove live cells. Then the supernatants were centrifuged at 2,000 × g for 20 min at 4 °C to remove dead cells. Next the supernatants were collected and pelleted after centrifugation at 10,000 × g for 45 min at 4 °C (Thermo Fisher Scientific) and resuspended in 1X PBS. Supernatants were filtered through a 0.22 µm membrane followed by centrifugation at 120,000 × g for 2 h at 4 °C (Thermo Fisher Scientific) to pellet the exosome-enriched fraction. The pelleted exosomes were resuspended in 1X PBS and collected by ultracentrifugation at 120,000 × g for 2 h at 4 °C. After isolation, the exosome pellets were finally dissolved in 1X PBS and stored at −80°C for further uses. Exosome isolation was done using kit reagent also.

Scanning electron microscopy (SEM)
Pellets containing EVs from healthy cells and senescent cells were fixed with 2% paraformaldehyde for 10 min. The samples were then added to cleaned silicon chips, followed by sonication in ethanol for 5 min and immobilized after drying under a laminar airflow. To make the surface conductive, a coating of 2-5 nm gold-palladium alloy was applied by sputtering (Argon as gas for plasma) before imaging by scanning electron microscopy (Inspect FEI, Netherlands). Analysis of EVs sizes was done using the SEM images via ImageJ software.

Nanoparticle tracking analysis (NTA)
Analysis of EVs concentration and distribution of hydrodynamic diameter was achieved by Nanosight NS300 (Malvern Technologies, UK). The samples were analyzed under various conditions, including cell temperature (at 25 °C), flow rate (= 50) and the duration of the recordings. For each measurement, the data were captured in five 30 s videos. Autofocus was adjusted to prevent indistinct particles. The data were analyzed using NTA 3.2 software with a detection threshold of 5.

Dynamic light scattering (DLS)
The particle diameter and size distribution of the isolated samples were measured by DLS instrument (ZetaSizer-HT Malvern). The Z-average values and the particle dispersion index (PDI) values were obtained for comparative study of the sizes of the isolated samples.

Cellular transfection and detecting uptake of exosomal HA-PTEN
A549 cells were seeded in antibiotic-free medium and incubated overnight prior to transfection. For transient transfection of HA-PTEN, Lipofectamine 3000 Transfection kit (Invitrogen) was used as per manufacturer's instruction (reagent: DNA = 2:1). After 24 h EVs were isolated from the media. PC3 cells were treated with these EVs for 4 h and 8 h, respectively. After that indirect immune labeling followed by fluorescence microscopy was performed to detect the transfer of HA-PTEN into PTEN mutant PC3 cells via exosomes (EVs).

Western blotting
Whole-cell lysates were prepared using cell lysis buffer (50 mm Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP-40, 1 mM dithiothreitol, 2 µg/ml aprotinin, 2 µg/ml leupeptin, protease and phosphatase inhibitor). The protein concentrations of the isolated exosomes and whole-cell lysates were also evaluated by Bradford assay. The cellular extracts and EVs were solubilized in a protein loading buffer, boiled for 5 min, and electrophoresed on a 10% SDS-polyacrylamide gel. Proteins were then transferred to methanol-activated polyvinylidene fluoride (PVDF) membrane. The membrane then is blocked in 5% non-fat dry milk in 0.05% Tween-20 in 20 mM Tris-Cl, pH 7.6 (TBST) for 1 h at room temperature. After overnight incubation with appropriate primary antibody, the membrane was washed with TBST followed by re-incubation with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. Proteins were detected by Advansta ECL western blotting detection reagent. The intensity of each band was measured by ImageJ software.

MTT assay
The inhibition of cell growth was measured by MTT assay. In brief, the human cell lines PC3 and WI-38, respectively, seeded in 12 well plates. PC3 cells was treated with EVs (100 µg/mL) isolated from mock-treated A549 and HeLa cells, respectively, and treated with EVs (100 µg/mL) isolated from A549 cells treated with H 2 O 2 . Similarly WI-38 cell was treated with EVs (100 µg/mL) isolated from mock treated and H 2 O 2 -treated A549 cells. In another set PC3 cells was treated with EVs (100 µg/mL) isolated from mock treated and H 2 O 2 -treated PC3 cells. MTT reagents are incubated for 3 h at 37 °C. The resulting formazan crystals were then dissolved in MTT solubilization buffer and the absorbance was taken with a UV-Vis spectrophotometer (Hitachi) at 570.

Senescence-associated (SA)-β-galactosidase staining assay
A549 cells were seeded overnight on cover slip. Next day, cells were washed with 1X PBS and were replaced with fresh medium. One set of cells were kept as control and the other set was treated with H 2 O 2 (150 µM) and incubated for 2 h. After the completion of the incubation period the cells were stained with a senescence-associated beta-galactosidase staining kit (Cell Signaling Technology, CAS 9860S). Finally, the stained coverslips were mounted on grease-free slides using 50% glycerol. Then, the slides were examined under a bright field of fluorescent microscope for detecting the senescent cells under 10 X magnification.

Fluorescence microscopy
A549 cells were seeded overnight on cover slip. The next day, cells were washed with 1X PBS and media were replaced by fresh medium. Then, one set of cells were kept as control and the other set was treated with H 2 O 2 (150 µg/ mL) and incubated for 48 h. The cells after being incubated were fixed with 4% paraformaldehyde solution for 15 min and permeabilized with 0.2% Triton X-100 at 4 °C for another 15 min. The cells were again washed followed by addition of 5% blocking solution (0.5% FBS in 1X PBS) for 1 h at room temperature followed by overnight incubation with H3k9me3 and γH2AX antibody in wash buffer (0.5% FBS and 0.05% Tween-20 in 1XPBS) at 4° C. Next day, the cells were washed with wash buffer and followed by addition of anti-rabbit IgG conjugated with FITC and antimouse IgG conjugated with Texas Red antibodies for 1 h at room temperature (dark condition). After washing, cells containing coverslips were mounted with the mounting solution containing DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, USA) and examined under a fluorescence microscope (100X objectives, Leica, Germany).

Comet assay
PC3 cells were seeded in 35 mm cell culture plates overnight. Next day, keeping one set without being treated and other two sets were treated with EVs derived from mocktreated and H 2 O 2 -treated A549 cells, respectively, for 48 h. Then, the cells were trypsinized and centrifuged. The pellets were washed in 1X PBS. PC3 cell pellet was mixed with 37 °C 0.8% low melting point agarose and transferred to normal melting point agarose-coated slides. The slides were mounted with cover slip and incubated at 4 °C for solidification. Then, the slides were immersed in lysis buffer (2.5 mM NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO, and 1% Triton X-100) for 1 h at 4 °C. After washing with neutralizing buffer (0.4 M Tris, pH 7.5), the slides were immersed into electrophoresis buffer within an electrophoresis tank. Electrophoresis was carried out at 25 V and 300 mA for 20 min. After which, the slides were washed with a neutralizing buffer for 40 min. After washing the slides were stained with EtBr solution and kept in the dark for 20 min. The stained slides were washed with 1X PBS and were finally mounted with cover slips using 50% glycerol as mounting media and scored using a fluorescence microscope (Leica, Germany). % of tail DNA (DNA fragment percent in the tail) was scored with the help of Komet Assay Software 5.5 at 40X magnification.

Cell damage detection a) Analysis of nuclear morphology by DAPI staining
Cells were seeded on cover slips. Next day, keeping one set as control, the other set cells were treated and then incubated for 48 h. Then, the cells were washed with 1X PBS for 2-3 times and the cell containing coverslips were mounted on grease-free slides with DAPI stain and finally observed under 100 X objective of fluorescent microscope.

b) Cell staining with Hoechst dye
Cells were seeded on cover slips. Next day, cells were treated and incubated for 48 h keeping one set as control. The cells were washed with 1X PBS for 2 to 3 times. Then the cells were stained with 1X Hoechst dye solution, followed by 15 min of incubation in dark condition, at room temperature. After washing with 1X PBS for 1 to 2 times, cells were mounted with 50% glycerol on grease-free slides and were examined under fluorescent microscope at 100 X magnification.

Cell cycle analysis
PC3 cells were seeded in 60 mm cell culture plates overnight. At 24 h after seeding, keeping one set without being treated and other two sets were treated with 100 µg/mL of EVs derived from mock-treated and H 2 O 2 -treated A549 cells, respectively, for 48 h. Then, the cells were trypsinized and centrifuged resulting in the formation of pellets. The pellets were washed with 1X PBS and fixed with chilled 70% ethanol and kept for overnight at −20 °C. Prior to stain with 50 µg/mL propidium iodide (PI, Invitrogen), the cells were incubated for 30 min with 100 µg/mL of RNase A (SRL, India) at 37 °C. The cell cycle was analyzed with flow cytometry.

Statistical analysis
A student's t-test was used to calculate the statistical significance of changes between the groups. P< 0.05 and 0.005 were considered as statistically significant. Data analysis was performed using the Origin Pro v.8 software (Origin Lab).

Characterization of EVs by SEM, NTA, and DLS
The size of EVs derived from mock-treated and H 2 O 2 -treated A549 cells were investigated using FESEM, as shown in Fig. 1a. The particle size of the EVs isolated from mocktreated cells was observed to be 60 ± 10 nm, while the EVs isolated from cells treated with H 2 O 2 showed slight increase in size which is 85 ± 15 nm with no change in surface morphology in both the cases. Additionally, the size distribution study by NTA and DLS also revealed a slightly larger size of EVs isolated from H 2 O 2 -treated cells as shown in Fig. 1d and b and c. Additionally, the purity of the exosome was analyzed by western blotting as shown in Fig. 1e. Presence of CD9, Flotillin 1, Annexin V, and CD63 indicated the purity of isolated exosomes (EVs).

Cell survivability
Effect of exosomes (EVs) on cell growth was explored on human prostate carcinoma cell lines PC3 and SV40-transformed normal lung fibroblast cell line WI-38. Survivability percentage of PC3 cells was calculated after treating them with exosomes (EVs) derived from mock-treated A549 cell line or HeLa cells at different time intervals (Fig. 2a  and b). For both the cases survivability of PC3 cells significantly reduced (*P < 0.05) to almost 80% after 24 h and 48 h intervals. But exosomes (EVs) derived from mock-treated and H 2 O 2 -treated PC3 cells when treated with PC3 cells itself, no change in survivability was observed in both the cases (Fig. 2c). Also, when normal fibroblast cell line WI-38 was treated with exosomes (EVs) isolated from mock-treated or H 2 O 2 -treated A549 cells, survivability of WI-38 cells remained the same (Fig. 2d). Thus the exosomes (EVs) secreted by A549 cells contain some factors which selectively inhibit growth of PC3 cells. Finally, when PC3 cells were treated with exosomes (EVs) isolated from H 2 O 2 -treated (150 µM, for 2 h) A549 cell line at different time intervals, the survivability significantly reduced to 60% (*P< 0.05, **P< 0.005) up to 48 h, and after that the cell began to restore its growth indicating exhaustion of growth inhibitory effect of exosomes (EVs) (Fig. 2e). Linear graphical representation and c columnar graphical representation of the A549 cell-derived EVs. Further DLS was performed to measure the average hydrodynamic particle sizes of the EVs isolated from mock-treated and H 2 O 2 -treated cells. The hydrodynamic radius (Rh) of the EVs was found to be 150 ± 10 nm and 200 ± 15 nm, respectively (c). Particle size obtained via DLS (average particle size) is much higher than that obtained via FESEM (actual particle size) due to DLS was performed in an aqueous medium, whereas FESEM was performed in a dry state. d NTA was performed to measure the concentration and distribution of hydrodynamic diameter of the EVs isolated from mock-treated and H 2 O 2 -treated cells. The hydrodynamic diameter of the EVs was found to be 149.3 ± 09 nm and 167 ± 12 nm, respectively. e Western blot analysis of cell lysate and A549-derived EVs for detection of exosomal marker proteins, which showed the expression of CD9, Flotillin1, Annexin V, and CD63

Detection of PTEN in PC3 cells treated with A549 cell-derived exosomes (EVs) by western blotting
Since aggressive nature of PC3 cells are due to the nonfunctional PTEN, we expect that exosomes (EVs) mediated delivery of PTEN may be responsible for their growth arrest. To prove this, we immunoblotted exosomes (EVs) content from both mock-treated and H 2 O 2 -treated A549 cells with PTEN antibody. We found the presence of PTEN in both the cases (Fig. 3a). In order to check the active function of PTEN transported by exosomes (EVs) in PC3 cell signaling pathways, we explored the signaling pathway  . 3 a Western blot analysis of PC3 cells treated with exosomes (EVs) isolated from wild type and H 2 O 2 -treated A549 cells for presence of PTEN protein, using GAPDH as control and for detecting the activity of PTEN by determining the presence of p-Akt, using Akt as control. Band intensity was measured by ImageJ software. Con-trol is normalized to 1. bWestern blot analysis of A549 cell-derived exosomes (EVs) for detection of HA-PTEN. c Fluorescence microscopic detection of HA-PTEN transfer into PC3 cells via A549 cellderived exosomes (EVs) at different time points associated with PTEN activity. Activation of PTEN resulted in dephosphorylation of Akt rendering the growth arrest as seen in Fig. 3a. In PC3 cells after exposure to exosomes (EVs) derived from mock-treated or H 2 O 2 -treated A549 cells, p-Akt is reduced compared to untreated cells. Furthermore, PTEN was detected when the PC3 cell was treated with exosomes (EVs) but no PTEN was detected in PC3 cell extract without any exosomes (EVs) treatment. Additionally, to track the PTEN transfer through exosomes (EVs), PC3 cells were treated with exosomes (EVs), derived from HA-PTEN-transfected A549 cells. Both western blot Fig. 3b and fluorescence microscopy Fig. 3c results showed successful transfer of HA-PTEN from A549 cells to PC3 cells via exosomes (EVs). Thus, exosomes (EVs) mediated delivery of catalytically active PTEN to PC3 cells was confirmed.

Cell cycle study
Cell cycle arrest may be mediated through DNA damage which may lead to apoptosis. Thus, we first analyzed the cell cycle of PC3 cells after exosomes (EVs) treatment. As seen in Fig. 4a the % of sub G1 cells did not increase, indicating the absence of apoptotic cells, while the % of S phase cells significantly decreased (*P < 0.05) from 31.97 to 10 and 6.16, respectively, in case of PC3 cells. Figure 4b and c represents the bar graph of cell cycle and quantification of number of cells in each phase, respectively.
To assure that the cell cycle arrest in the PC3 cells were mediated via PTEN only and to confirm no involvement of another major tumor suppressor protein TP53 in this phenomenon, western blotting was performed. The results of western blotting Fig. 4d showed absence of TP53 in A549 cell-derived exosomes (EVs), showing presence of the same in the cell lysate of A549, strongly confirmed the PTENmediated cell cycle arrest in PC3.

Comet assay
Growth arrest of PC3 cells may be mediated by DNA damage induced by the factors present in the exosomes (EVs). To address this, we performed comet assay and observed the DNA damage repair activity of PTEN in the PC3 cells treated with exosomes (EVs) isolated from mock-treated and H 2 O 2 -treated A549 cells. DNA damage was observed in those cells without any exosomes (EVs) treatment and exosome-treated PC3 cells showed no damage (Fig. 5a). The amount of damage was determined by % of tail DNA. For control PC3 cells, cells treated with exosomes (EVs) derived from H 2 O 2 -treated A549 cells and PC3 cells treated with exosomes (EVs) derived from mock-treated A549 cells, the % of tail DNA was significantly found to be 18%, 8.67%, and 11%, respectively, as shown in Fig. 5b (**P < 0.005).

Analysis of cellular senescence
Thus functional PTEN delivered by exosomes (EVs) mediates temporary growth arrest in PC3 cells. Now both mocktreated and H 2 O 2 -treated cells derived exosomes (EVs) can induce growth arrest in PC3 cells. We only found a little larger exosomes (EVs) in H 2 O 2 treatment. Thus, we wanted to determine the fate of H 2 O 2 -treated A549 cells. In simple DAPI and Hoechst staining of A549 cells no change in  nuclear morphology was observed ( Fig. 6a and b). Since no apoptosis was observed in A549 cells after H 2 O 2 treatment (data not shown) the possibility of senescence in A549 cells after treatment with H 2 O 2 was explored. Senescenceassociated beta-galactosidase assay (Fig. 6c) showed induction of senescence in A549 cells after treatment with H 2 O 2 (150 µM) for 2 h. Quantitatively, the % of senescent cells significantly increases (*P< 0.05) (Fig. 6d) in H 2 O 2 -treated A549 cells at 2 h (56.75 ± 3.14) compared to mock-treated A549 cells (7.6 ± 1.32). Additionally, senescence-associated heterochromatin foci were also tested for H 2 O 2 -treated cells by observing foci of H3K9me3 (Fig. 6e) and γ-H2AX foci (Fig. 6g). Here we also observed ( Fig. 6f and h)

Discussion
Exosomal cargo contains numerous factors, including DNA, proteins, and RNA [3]. Here we have seen that exosomes (EVs) from lung fibroblast cells can induce growth arrest in PC3 cells. PC3 cells are implicated as deleted mutations in PTEN and lack of PTEN are supposed to be responsible for its abnormal growth [14]. Thus, delivery of functional PTEN may be a potential therapeutic approach to arrest growth of cells having downregulated PTEN. Exosomes (EVs) mediated delivery of different agents are emerging as a potential approach as it is nonimmunogenic [4,15]. Here we have seen the exosomes (EVs) derived from A549 cells and HeLa cells are capable of inhibiting growth of PC3 cells. While most of the studies indicated the tumor-promoting activity of exosomes (EVs) secreted by various cells [1,7], some reports available where exosomes (EVs) seem to inhibit tumor growth [16]. The protein composition of exosomes (EVs) is not unique and remains to be explored further. But the content of the exosomes (EVs) varies between cells from which they originate proteins which are no longer necessary for their survival or excess protein production due to mutation [17]. Thus, exosomal content was suggested to be biomarkers of different diseases. Although, due to their endosomal origin they contain some marker proteins (flotillin, CD9, CD63, etc.) which are found in exosomes from different cells [1]. Here also we have seen the presence of some of these proteins in our exosome. Here, at first, we found that exosomes (EVs) from lung cancer cells can inhibit the growth of prostate cancer cells specifically and have Values are the mean ± SD of three independent experiments. **P < 0.005 no effect on normal lung fibroblast. Also exosomes (EVs) from prostate cancer cells (PC3) have no effect on PC3 cells. We initially thought that under stress conditions lung cancer cells may act differentially and thus can change the exosomal (EVs) content and impart different effects on PC3. But even under stress conditions, exosomes (EVs) secreted by A549 cells can arrest the growth of PC3. Since some miRNA carried by exosomes (EVs) may alter the DNA damage response pathway [18], we explored the possibility also and found that was not the case. Induction of apoptosis by exosomal (EVs) content is also reported [14] but in our case, the exosomes (EVs) cannot induce apoptosis in PC3 cells. Genetic data reveal that PC3 cells have deleted PTEN where lung cancer cell A549 has mutated Ras protein [14,19]. This prompted us to look for PTEN in the A549-derived exosomes (EVs). The possibility that TP53, another major tumor suppressor protein could cause the growth arrest in the PC3 cells was abandoned as no TP53 was found in A549 cell-derived exosomes (EVs) in our western blot result. Surprisingly even under stress conditions, lung cancer cells can secrete exosomes (EVs) containing PTEN. Functional analysis of PTEN was also carried out. The most potential approach in this case is to analyze the PI3K pathway, where we found delivered PTEN in PC3 cells can inhibit the phosphorylation of Akt. Usually in cancer cells Akt is hyperactivated by phosphorylation and induces tremendous cell growth even under DNA damage conditions rendering genomic instability [20]. In our case we also found dephosphorylation of Akt in PC3 cells indicating functionally active PTEN was delivered and Akt-mediated signaling for the growth of PC3 cells is perturbed. It has been reported earlier that cancer cells use a noble mechanism in the downregulation of PTEN [21][22][23]. It seems likely that A549 cells while having wild-type PTEN may use exosomal pathways to overcome the tumor suppressor activity of PTEN. Thus functional PTEN, even it is present in lung cancer cells, may not impose growth arrest activity on A549 cells. On the other hand, senescence induced by H 2 O 2 in A549 cells resulted in excretion of PTEN and must be accompanied by some other factor associated with small extracellular vehicle (sEV) responsible for changing several activities of recipient cells. In our case we have seen larger volume of sEV compared to untreated A549 cells indicating the amount of content increases under senescence condition (Fig. 1a, c, and d, respectively). But the growth arrest is temporary as after 72 h of treatment the inhibitory effect vanishes perhaps due to exhaustion of imported PTEN. Additionally, PTEN is emerging as a DNA repair protein and responsible for maintaining genomic stability. In our study we have seen that exosomes (EVs) treated PC3 cells have less amount of tail DNA in comet assay (Fig. 5b). Thus, A549 cells derived from the exosomes (EVs) contain functionally active PTEN which is likely to be responsible for growth inhibition of PC3 cells. These findings have important therapeutic implications to treat prostate cancer cells or any cancer associated with dysfunction PTEN. To observe the change in nuclear morphology after treating the exosomes (EVs) donor A549 cell with H 2 O 2 we used staining procedures with DAPI and Hoechst dye. In DAPI staining, the characteristic change in nuclear morphology was observed in cells treated with H 2 O 2 while the nucleus of the mock-treated cells appeared intact (Fig. 6a). The heterochromatin foci which are observed in case of senescent cells due to staining with Hoechst dye was detected in H 2 O 2 -treated A549 cells and were absent in mock-treated A549 cells (Fig. 6b). Exosomes (EVs) produced from H 2 O 2 -treated lung cancer (A549) senescent cells transfer PTEN in PTEN-null (PC3) cells resulting in cell growth arrest as long as 48 h. PTEN, if inactivated selectively and temporarily induces hyperactivation of signaling pathway, leading to cellular senescence as reported by Alimonty et al. [24]. Exosome (EVs) is also a constituent of senescence-associated secretory phenotype (SASP) along with a multitude of proteins, such as growth factor and inflammatory cytokines [24]. Cell survivability in PTEN-null cells (PC3) increases after 48 h which may be concluded by the fact that PTEN derived from exosomes (EVs) of senescent cells may be selectively inactive which, on meeting the growth factor present within exosomes (EVs), induces cell survivability. The key point is H 2 O 2 treatment resulted secretion of PTEN among other factors which are released by senescencent cells. While other factors may have inflammatory cytokines but we have focused on PTEN in H 2 O 2 -treated A549 cells. PTEN, being a well-known DNA repair protein, which [25] reduces the DNA damage and exerts its Cell cycle arrest property to PC3 cells. Here we analyzed the exosome (EV)-mediated PTEN secretion by H 2 O 2 -treated A549 cells reduces the DNA damage and induces Cell cycle arrest at G1/S phase of PC3.

Conclusion
From our study it can be concluded that exosomes (EVs) released from senescent cells can express PTEN and it also delivers PTEN into PTEN-null cancer cells. Expression of PTEN in the cancer cell can suppress the proliferation by G1 cell cycle arrest. Therefore, this study can open an anticancer strategy by improving the functional dose of PTEN.