Cell Lineage Dependent Apoptotic and Immunomodulatory Signaling Induced By Parthenin Analog P19: An Insight to Molecular Mechanism and Therapeutic Implications

Leukemia is one of the deadliest types of cancer. Specically, acute lymphoid leukemia (ALL) has been considered as one of the most lethal types of cancer. Due to the rapid progress in the various cancer cases and the accumulation of resistances in the cancer cells, the discovery of the new lead molecules with more effective anticancer properties are required. There is a growing interest in using herbal products/analogs as multi-component agents (as anticancer agents and immunomodulators) for cancer treatment. In the present investigation, an attempt has been made to explore the anticancer and immunomodulatory activity of P19 in ALL. P19 was reported to demonstrate the anticancer activity by activation of apoptotic signaling events through robust NO formation in human leukemia HL-60 cells. Contrary to this observation, P19 mediated apoptosis in Raji cells was independent of NO and ROS induction. The mechanism of P19 was observed to be cancer cell lineage dependent. Further, P19 demonstrated very effective anticancer properties against ALL (IC 50 3µM). Molecular investigations revealed that P19 induced mitochondrion mediated apoptosis by Bax localization to mitochondria and enhanced cytosolic calcium in the cytoplasm. Further activation of the caspase 3, caspase 8 and PARP cleavage suggested the involvement of the caspase-mediated apoptosis. Anti-proliferative activity revealed the telomerase inhibition and cell cycle arrest in G 0 /G 1 phase after P19 treatment. Immunomodulatory effects of the P19 revealed the enhanced INF (cid:0) and NO production in Jurkat and THP cells. Owing to its antiproliferative and immunomodulatory potential against leukemia cells P19 can further be explored as an effective therapeutics against leukemia. melting curve analysis. Beta-actin was considered as an internal control. Data are presented as the mean ± S.D. of three similar experiments(C, D) FACS analysis histograms of anti IFNγ antibody stained Jurkat cells (C) and THP-1 cells (D) after P19 treatment. Jurkat and THP-1cells were treated with indicated concentrations of P19 (below and at IC50 ) and analyzed using ow cytometry after staining with uorochrome-conjugated anti IFN (cid:0) antibody. Mean values represent the change in expression of IFN (cid:0) expressed as mean uorescence intensity. Five thousand events were counted per tube. (E, FACS analysis histograms of NO estimation in Jurkat (E) and THP-1 (F) cells after P19 treatment. Jurkat and THP-1 cells were treated with indicated concentrations of P19 (below and at IC50 ) for 48 h followed by DAF-2-DA staining and ow cytometric analysis. Mean values indicate NO stress inside the cells. H) RT-PCR analysis for iNOS expression in Jurkat, and THP-1 (G) cells after treatment with P19. Western blot analysis of iNOS expression and nuclear localization of NF-κB in Jurkat (I) and THP-1(J) cells after P19 treatment at indicated concentrations. Total cell lysates or cell lysate after cytosolic and nuclear fractionation were resolved on 10% SDS-PAGE for immunoblot analysis of iNOS and NF-κB expression. P19 treatment induced enhanced iNOS expression and nuclear localization in Jurkat and THP-1 cells. Untreated cells were considered as control.* represents the statistically signicant (P<0.05) difference between control and P19 treated cells. Data are representative from one of three similar experiments.


Introduction
Cancer is an uncontrolled growth of cells. The primary treatment options available are chemotherapy and radiotherapy. One of the major drawbacks of chemotherapy is that the agents used exhibit signi cant cytotoxicity or side effects against normal cells [1]. Another drawback of chemotherapy is that cancer patients acquire resistance to traditional chemotherapeutic agents [2]. Therefore, it is necessary to search and develop new compounds that provide suitable speci c antiproliferative effects that can be developed as anticancer agents. The major potential source for anticancer drug discovery is the diverse plant ora. Natural resources are routinely consumed by animals and exhibit lesser cytotoxicity against normal cells.
To date, many chemotherapeutic drugs have been obtained from the plants as direct agents or as lead molecules [3]. Recent trends in research included the focus on the lid molecules, which along with the anticancer also possess the immunomodulatory nature. Recently a plant derived compound zerumbone has been shown to induce both anticancer as well as the immunomodulatory effect [4].
Parthenium hysterophorus(Asteraceae), a weed also known as congress grass, is an annual herb. Plant has been used as a folk remedy for the treatment of infectious and degenerative diseases [5]. To enhance its bioactivity, analogs of parthenin were synthesized by substitutions at different reaction centers to establish a structure-activity relationship [6]. A novel parthenin analog P19, was reported to demonstrate anticancer activity by activation of apoptotic signaling events through robust NO formation in human leukemia HL-60 cells [5]. The pro-apoptotic activity of P19 suggested its potential use as a promising anticancer therapeutic. However, its immunomodulatory activity has not been studied to date, as the response of several anticancer drugs was observed to be different in different types of cancers [7]. Therefore, an attempt has been made to investigate the anticancer potential and immunomodulatory activity of P19 against ALL (acute lymphoid leukemia). Our studies suggested that P19 acts as a dual agent by inducing both apoptotic as well as immunomodulatory activities.

Materials And Methods
Cell culture, growth conditions and treatment Raji cell line (B cell lymphoma), Jurkat (T cell lymphoma), THP-1 (monocytes) were procured from the National Center for Cell Science (NCCS) Pune, India. Raji cells were grown in RPMI-1640 medium containing 10% FCS (Himedia India), 100 units penicillin/100 µg streptomycin per ml medium in CO 2 incubator at 37 0 C with 95% humidity and 5% CO 2 gas environment. Monocytes were differentiated to macrophages by using PMA (5 ng/ml) for 48 h [8]. Cells were treated with P19 dissolved in DMSO while the untreated cultures received only the vehicle (DMSO, <0.2%, v/v).

Acridine orange/ethidium bromide staining and SEM imaging
To study the nature of P19 induced cytotoxicity, Raji cells were grown in the presence of 1.0 μM, 3.0 μM of P19 for 48 h, and were observed under uorescence microscope (Nikon) after staining with acridine orange/ethidium bromide stain (10 μg/ml). Cells stained with the dual staining were considered as the apoptotic cells. Scanning electron microscopy (SEM) was carried out using previously de ned methods [10].
Cell cycle analysis and chromosome preparations Cell cycle analysis was carried out using propidium iodide, as described previously [9]. Brie y, 1x10 6 cells/ml after P19 (1.0 µM, 3.0 µM) treatment for 48 h were washed with chilled PBS twice. Further cells were xed with 70% ethanol for 40 min. After that, cells were rewashed with PBS and resuspended in 500 µL of staining solution containing propidium iodide, detergent, and RNAse followed by ow cytometric analysis. Raji cells (1×10 6 cells/ml) were seeded and treated with P19 (1.0 µM, 3.0 µM) for 48 h for chromosome preparations. Both P19 treated and untreated cells were subjected to colcemid treatment (0.1µg/ml) for 2h before terminating experiment. G-banding was done using 0.005% trypsin, and chromosomes were analyzed under a light microscope. Comparative mitotic indices of the slides were calculated by considering the average values of the ratio of metaphase plate vs. the number of cells in ve different areas of slides at 10× magni cation.

Telomerase extract preparation and Conventional trap assay
The telomerase extract preparation and conventional trap (telomerase repeated ampli cation protocol) assay was carried out by the method of Mender and Shay, 2015 with minor modi cations [11].
Control and P19 treated cells were used for the telomerase extract preparation. Raji cells (1 x 10 6 / ml) with/without treatment were washed once in PBS and pelleted at 5000 rpm for 5 min at 4 0 C. Further, cells were resuspended in 500 µl of ice-cold washing buffer (10 mM HEPES-KOH (pH 7. RT-PCR and quanti cation of mRNA levels Total RNA was isolated from the treated and untreated cells after P19 treatment using the trizol reagent (SigmaAldrich). An equal amount of RNA was used to synthesize cDNA after DNAse treatment using the Revert Aid rst-strand cDNA synthesis kit (thermo scienti c). RT-PCR (Real time PCR) and semi quantitative reverse transcription PCR was carried out to check the alteration in the level of mRNA expression. Real time PCR analysis was performed in the eppendorf real-plex system using the SYBR Green PCR Master mix (Thermo scienti c USA). Real time PCR was carried out for p21, IFN-γ, IL-2, and βactin using gene speci c primers. β-actinserved as an internal control. The speci city of PCR products was analyzed using the melting curve analysis, and the delta CT method was used to quantify alteration in expression [12]. Expression of hTERT, hTR, TP-1, Baxand Bcl2 using the semi-quantitative reverse transcription PCR as described previously [13].
Flow Cytometry based analysis Raji cells (1.0 x 10 6 cells/ml) were treated with P19 for 48 h and washed once in PBS. The washed cells were further, incubated with dyes 2'-7'dichloro uorescin diacetate (DCFH-DA), 4,5-diamino uorescenediacetate (DAF-2-DA), JC-1, Fluo-3 AM and subjected to Reactive oxygen species (ROS), nitric oxide (NO), mitochondrial membrane potential and cytosolic calcium detection as described previously [5,14]. For uorochrome-conjugated antibody staining, the Raji, Jurkat and THP-1 cells were washed with PBS. Further cells were xed and permeabilized using the Cyto x/Cytoperm kit (BD, Bioscience, USA) for 20 minutes on ice. Cells were pelleted, rewashed with Perm Wash Buffer (BD, Bioscience, USA), and stained with uorochrome-conjugated anti IFNγ, anti p21 antibodies (Santa Cruz Biotechnology, USA), at 4 0 C for one hour. Finally, cells were washed again and resuspended in the Perm Wash Buffer for ow cytometry analysis. Flow cytometric analysis was performed on a BD FACS Canto II (BD Biosciences, USA) for a maximum cell count of ten thousand and analyzed using BD FACSDiva software.
Beta-Galactosidase staining and in gel activity assay Beta-galactosidase staining was performed as described previously [15]. Brie y, Raji cells were treated with P19 for 48 h, followed by washing in PBS. After thiscells were xed in 2% formaldehyde/0.2% glutaraldehyde and rewashed with PBS at 37°C. For staining cells from the previous step were incubated with fresh senescence-associated β-Gal stain solution.For in gel activity assay of catalase and superoxide dismutase (SOD) total cell lysate (equivalent to one unit of either catalase or SOD activity) was subjected to native polyacrylamide gel electrophoresis at room temperature. Afterward, gels were rinsed with 200 ml water and then stained for either catalase or SOD activity using the protocols described previously [16].

Results
P19 induced anti proliferative and apoptotic effect on leukemic cells P19 inhibited Raji cells in a dose dependent manner with IC 50 of 3.0 ± 0.60 (µM) (Fig. 1A). The effect of P19 on THP-1 and Jurkat cells was studied as these cells were further used to nd out the immunomodulatory effects. P19 decreased the proliferation of Jurkat and THP-1 cells with an IC 50 of 1.0 ± 0.3 (µM) and 3.0 ± 0.4 (µM), respectively ( Fig 1A). To con rm the nature of cytotoxicity, Raji cells were treated with P19 (1.0 µM, 3.0µM) and subjected to DNA laddering assay, acridine orange/ethidium bromide staining. P19 treated Raji cells revealed markedly increased fragmented DNA in comparison to untreated cells ( Fig. 1 B). Furthermore, acridine orange/ethidium bromide double staining revealed large green nuclei with intact membranes in untreated cells. However, P19 (3.0 µM) treated Raji cells revealed a signi cant decrease in large green nuclei and exhibited signs of apoptotic cells indicated by orange and red uorescent (Fig. 1 C). Further morphological analysis P19 treated Raji cells using SEM revealed characteristic membrane blebbing while untreated cells revealed intact membrane indicating the cytotoxic effect of P19 ( Fig. 1 D). Raji cells treated with P19 were subjected to propidium iodide and observed under the uorescent microscope. P19 treated cells at 3.0 µM showed a signi cant condensation of the nucleus, while the untreated cells showed an intact nucleus ( Fig. 1 E). Collectively these results indicated that Raji cells underwent apoptosis after treatment with P19.
P19 caused cell cycle arrest in G 0 /G 1 phase and did not contribute to genomic instability Cell cycle analysis revealed that P19 (1.0 µM, 3.0 µM) treatment to Raji cells induced cell cycle arrest in G 0 /G 1 phase. P19 treated Raji cells revealed increased count from 54% to 72% in G 0 /G 1 phase in comparison to control (untreated cells), while a signi cant decrease in S phase and G 2 /M was observed suggesting G 0 /G 1 phase arrest in Raji cells ( Fig. 2 A, B). Further to analyze the effect of P19 on genomic stability, chromosome preparation was carried out after P19 (1.0 µM, 3.0 µM) treatment. P19 treated Raji cells did not show any chromosome breakage as compared to control cells (Fig. 2 C). The comparative mitotic index of control slides was found to be 0.35, while for P19 treated slides, 0.17 was observed. Collectively these observations suggestedthat P19 induced apoptosis in Raji cells was independent of genomic instability.
P19 mediated apoptosis independent of NO and ROS in Raji cells.
Since P19 was documented previously to induce ROS mediated apoptosis in HL-60 cells, we evaluated this molecule for its potential to induce ROS and NO in Raji cells [5]. P19 treated HL-60 cells were considered as positive control (Fig 3 A, B). We did not observe any signi cant variation in ROS and NO content in P19 (1.0 µM, 3.0 µM) treated Raji cells, whereas P19 treated HL-60 cells showed a marked increase in ROS and NO ( Fig. 3. A, B). Further to rule out the role of catalase and SOD in P19 treated Raji cells, in-gel activity was accessed. No signi cant change in the activity of SOD and catalase was observed (Fig. 3 C). The activity pro le correlated with the transcription pro le of these genes. Semi quantitative reverse transcription PCR analysis revealed no signi cant change in the expression of these genes (Fig 3 D). Collectively these results suggested that P19 did not affect the antioxidant system and P19 mediated apoptosis was independent of ROS and NO induction in Raji cells.

P19 enhanced cytosolic calcium level and induced mitochondrion mediated apoptosis
The calcium ion is an important secondary messenger in controlling cell signaling and cell death [17].
Enhanced cytosolic calcium level was documented previously linked to mitochondrial and caspase mediated apoptosis induction [18]. Therefore, evaluation of cytosolic calcium was carried out after treatment with the P19. Results revealed the enhanced cytosolic calcium after P19 treatment in Raji cells as compared to control (Fig. 4 A). Simultaneously treatment of Raji cells with P19 resulted in upregulation of Bax and downregulation of antiapoptotic Bcl2 protein (Fig 4 C). Both cytosolic calcium and Bax translocation have been documented to induce mitochondrial mediated apoptosis [19]. Therefore, to determine the translocation of Bax from the cytosol to mitochondria, cytosolic and mitochondrial fractions from P19 treated Raji and control cells were subjected to western blot analysis. A signi cant increase in the level of Bax in the mitochondrial fraction was concomitant with a decreased level of Bax expression in cytosolic fraction in P19 treated cells as compared to control (Fig 4 D).
Simultaneously an alteration in mitochondrial membrane potential and a signi cant increase in the cytosolic cytochrome c were observed after P19 treatment as compared to control (Fig. 4 E, F). These studies suggested that Bax could lead to mitochondrial membrane potential loss, and which can result in release of cytochrome c from the mitochondrion. Together these observations indicated that P19 treated Raji cells underwent apoptosis in the mitochondrial mediated pathway.
The induction of caspases and poly-ADP ribose polymerase (PARP) has been considered as a critical feature of apoptosis [20]. Therefore, after monitoring the enhanced expression of cytosolic cytochrome c, we analyzed the expression of caspase 3. Western blot analysis showed the enhanced level of cleaved caspase 3 in P19 treated Raji cells while an intact band of caspase 3 was observed in control cells (Fig 4  H). Further caspase 3 and caspase 8 activation was found linked with PARP cleavage. PARP cleavage leads to the activation of various endonucleases that cleave DNA and results in DNA fragmentation. The higher level of cleaved PARP was observed after P19 treatment in Raji cells. Collectively all these observations suggested that P19 induced caspase mediated apoptosis in Raji cells.
P19 induced TP53 upregulation and inhibition of telomerase activity TP53 participates in the cellular response to various stress conditions and plays a signi cant role in apoptosis induction [21]. Therefore, we evaluated the status of TP53 in P19 induced apoptosis. When Raji cells were treated with P19 (1.0 µM and 3.0 µM) a signi cant upregulation in TP53 protein was observed as compared to control (Fig.5 A). TP53 has been documented to activate the expression of p21 [22]. Therefore, we further analyzed the expression of p21 at both mRNA level (RT PCR) and protein level ( ow cytometry) (Fig. 5 B). It was observed that P19 treatment upregulated the p21 mRNA by more than 3 fold in Raji cells as compared to control. At the same time, ow cytometric analysis after staining with uorochrome conjugated anti p21 antibody revealed nearly 2 fold enhanced expression of p21 after P19 (3.0 µM) treatment (Fig. 5 B, C). These results suggested that P19 induced TP53, which in turn increased the p21 expression resulting in cell cycle arrest and apoptosis induction.
Inhibition of the telomerase activity is the key feature of the anticancer drug discovery [23]. Keeping this in mind we evaluated the effect of P19 on telomerase inhibition. TRAP assay revealed that telomerase activity was inhibited in a concentration dependent manner as indicated by a decrease in laddering pattern with an increase in the concentration of P19 (Fig 5 E). Further, Raji cells treated with P19 showed the most β-galactosidase staining as compared to untreated cells, which suggested that P19 mediated inhibition of telomerase lead to senescence induction in Raji cells (Fig 5 D). To determine the mechanism of action of P19 on telomerase, we evaluated the expression of three telomerase components, hTERT, hTR, and TP-1, using the semi quantitative reverse transcription PCR as described in the material and method section. The results revealed no signi cant change in the expression of any subunit as compared to control. These results suggested that P19 inhibited the telomerase by targeting it at protein level (Fig 5  F).

P19 modulates the expression of various cytokines
Since targeting cancer with a dual strategy (anticancer and immunomodulatory) could be a more effective treatment, we evaluated the immunomodulatory activity of P19. Interleukin 2 (IL-2) and interferon-gamma (IFN ) were selected for our investigation, since these molecules have been documented to play a signi cant role in anticancer activities [24]. The expression of cytokines (IL-2 and IFN ) was analyzed by RT-PCR. A signi cant upregulation in the expression of cytokine IFN (7 fold), IL-2 (6 fold) was observed in Jurkat cells after P19 treatment (Fig. 6 A). Similarly, in THP-1 cells (differentiated to macrophage cells), P19 treatment resulted in upregulation of IFN (5 fold), IL-2 (3 fold) (Fig. 6 B) in comparison to untreated cells. The results were further validated by staining of Jurkat and THP-1 cells with uorochrome conjugated anti IFN antibody (Fig. 6 C). P19 enhanced the expression of IFN in Jurkat and THP-1 cells. P19 could induce Jurkat cells more e ciently to produce IFN at a very low concentration (0.5-1.0 µM). These results suggested that P19 could modulate the immune system by enhancing the expression of major cytokines, including IFN . Because NO production is one of the critical mechanisms in the immune response we analyzed the level of NO in Jurkat and THP-1 cells [25]. Contrary to Raji cells, the treatment of Jurkat and THP-1 cells with P19 resulted in enhanced NO production (Fig. 6  E). As the NO production is dependent on iNOS (inducible nitric oxide synthase), the effect of P19 on the expression of iNOS was also checked. The induction in NO production was further supported by enhanced iNOS expression in Jurkat (Fig. G, I) and THP-1 cells (Fig. H, J). Further, to analyze the mechanism behind the simultaneous expression of various cytokines and NO induction, the nuclear localization of NF-κB was analyzed by westren blot analysis in Jurkat and THP-1 cell lines. NF-κB is the key transcription factor involved in the activation and transcription of various cytokines along with NO induction by the iNOS mediated pathway [26]. NF-κB was translocated to the nucleus after treatment with P19. It was observed that P19 caused cleavage of NF-κB and enhanced the nuclear localization of NF-κB in Jurkat and THP-1 (Fig 6 I, J) cells. These studies suggested that P19 induced NF-κB mediated immunomodulation.

Discussion
To treat the cancer more e ciently and overcome the accumulating drug resistance new anticancer molecules are still needed. Plant derived anticancer drugs originated from plants that caught the attention of the scienti c community due to their minimum side effects [27]. Several compounds based on plant origin have been identi ed for their anticancer potential [28]. These compounds generally target the genes involved in the progression of cell cycle/apoptosis [29]. In the present investigation anticancer potential of P19 was evaluated against Raji cells. P19 demonstrated a signi cant cytotoxic effect against Raji cells. The IC 50 of P19 for Raji cells is slightly higher than the previously reported for HL-60 cells [5].
Further, DNA fragmentation assay and microscopic analysis pointed towards apoptosis as the mechanism for observed cytotoxicity with P19 compound. Beside the induction of cytotoxic action in Raji cells, the compound induced the anti-proliferative effect as revealed by the cell cycle arrest in G 0 /G 1 phase and was independent of genomic stability or chromosomal damage. Some of the plants derived anticancer compounds such as curcumin formerly documented to induce genomic instability in Raji cells [9]. However, there was no change in chromosome integrity in P19 treated Raji cells.
P19 was reported to induce apoptosis through robust NO production in HL-60 cells [5]. Contrary to this observation, P19 mediated apoptosis in Raji cells was independent of NO and ROS induction. Further no change activity and gene expression in catalase and SOD ruled out the involvement of antioxidant defense mechanisms. The mechanism of P19 was observed to be cancer cell lineage dependent. These studies are supported by the previous studies where many natural compounds like Alternol works in a cell lineage dependent manner and follow different mechanisms in different cell lineages [30]. As reported earlier, P19 induced translocation of Bax to mitochondria, disruption of mitochondrial function with concurrent loss of mitochondrial membrane potential, release of cytochrome c to the cytosol, activate caspase cascade in HL-60 cells [5]. In concordance with these studies, Raji cells treatment with P19 revealed enhanced cytosolic calcium and translocation of Bax from cytosolic fraction to mitochondrial fraction. While the expression of Bcl2 decreased. Previously also many anticancer drugs involved in cancer treatment or under trial are very well known to induce Bax mediated apoptotic effects in malignant cells [31]. Bax is a pro apoptotic protein and documented to alter mitochondrial membrane potential [32]. Similarly altered mitochondrial membrane potential as well as enhanced cytosolic cytochrome c level was reported after P19 treatment in Raji cells. Recent studies both in vitro and in vivo revealed that various natural compounds exert their anticancer mechanism by mitochondrial mediated pathways. Similarly, our study also suggested that P19 treatment of Raji cells resulted in mitochondrial dysfunction and enhanced cytochrome c levels. Inductions of caspase and PARP cleavage have been considered as downstream executioners of enhanced cytochrome c levels [33]. In the same way P19 treatment also showed caspase 3, caspase 8 induction and PARP cleavage. All these studies together revealed mitochondria and caspase mediated apoptosis induction in Raji cells after P19 treatment.
TP53 is a major tumor suppressor that is activated in response to DNA damage. Downstream it activates the expression of p21 and can also target the telomerase [34]. Many potential strategies to target the cancer are documented to exert anticancer effects by restoring the TP53 mediated pathways [35]. Therefore, an attempt was made to study the expression pattern of TP53 and its downstream effector p21 after treatment with test compounds. P19 could induce the expression of TP53 and p21 in Raji cells. This might be a reason for cell cycle arrest in the G 0 /G 1 phase. We demonstrated that in Raji cells, telomerase activity was suppressed by P19 in a concentration manner. Previous studies have indicated that telomerase expression is associated with cell immortalization and tumorigenesis, which made telomerase an attractive target for cancer therapy [36]. Especially in leukemia where over-expression of telomerase has been reported earlier [37]. Recent study also suggested that anticancer effects of drugs can be enhanced by inhibiting telomerase activity [38]. Few Parthenin compounds have also been documented to suppress the telomerase activity in cervical cancer cell lines [6]. Our studies also suggested that P19 inhibited the telomerase activity in a concentration dependent manner, while no change in the expression of telomerase subunit was observed. These studies were further supported by the activation of senescence in Raji cells after P19 treatment, as demonstrated by beta-galactosidase assay. Studies with P19 on Raji cells collectively suggested that P19 could lead to the cell cycle arrest and inhibit the telomerase activity.
The immune system's natural capacity to detect and destroy abnormal cells plays a vital role in cancer prevention. Cytotoxic drug treatment and tumor microenvironment conditions induce several alternative modes for cancer cell death. However, certain forms of drug-induced cell death are much more useful than others because of their ability to activate potent anticancer immune responses [39]. Immunotherapy is another type of cancer treatment involving modulation of the patient's immune system for targeting cancer cells. Some of the anticancer drugs (such as taxol) were reported to exert an additive effect in the treatment of cancer by the modulation of the host defense system [40]. Among the immunomodulatory effects, NO production by immune cells is a crucial event. It has been reported that NO derived from macrophages, kupffer cells, natural killer cells, and endothelial cells participates in tumoricidal activity against many types of tumors [41]. Along with NO, cytokines, IL-2 and IFN play a signi cant role in ghting against the cancer cells. Among these, IL-2 has been considered as the rst effective immunotherapy against the tumors [42]. Dunn et al., 2006 have shown that immunotherapy with IFN in cancer patients improved the lives of cancer patients [43,44]. Therefore,the study was extended to reveal the immunotherapeutic potential of P19. Upon treatment with P19 Jurkat and differentiated macrophages lead to signi cant production of IFN , IL-2. Macrophages and Jurkat cells generated a large amount of NO, whereas in the case of Raji cells no ROS or NO production was observed. The enhanced level of iNOS suggested an iNOS mediated pathway involvement in NO production. Recent research has shown that cytokines (IL-2, and IFN ) and iNOS can be induced by NF-κB mediated pathway [45,46]. The treatment of Jurkat and THP-1 cells with the P19 followed by nuclear localization of NF-κB suggested the involvement of NF-κB in the induction of cytokines and iNOS, resulting in the production of NO. These macrophages, in turn, play an important role in immune surveillance against tumors during their development by presenting tumor antigen to cytotoxic T cells and releasing tumoricidal substances like cytokines and nitric oxide. Previously, many natural plant products like garlic and curcumin have also been shown to modulate the immune system [47,48]. A comparison of P19 to other molecules suggested that P19 is a novel anticancer molecule that possesses cytotoxic properties and immuno-modulatory functions even at very low dose kinetics.
Conclusion P19 inhibited cancer cell proliferation. It inhibited telomerase activity of these cells and induced TP53, which in turn increased the p21 expression resulting in cell cycle arrest and apoptosis induction. P19 also induced the expression of IL-2, IFN in macrophages, and T cells via activation of NF-kB, resulting in the induction of iNOS and the NO. In this study, we showed that in addition to cytotoxic activity, P19 exhibited immunomodulatory activity at a relatively low dose. These observations suggested that P19 could be a promising anticancer agent to consider for more research on animal tumor models and even human clinical trials Declarations Acknowledgement We thank Dr. SC Taneja for providing the P19 test compound.

Availability of data and materials
All data analyzed in this study are included in this published article.

Author contributions
Vishal Sharma performed experiments and wrote manuscript. Ajay Kumar participated in data analysis. Jagdeep Kaur supervised study and designed experiments.

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Ethic approval is not required.

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Funding
This study was supported by the Council of Scienti c and Industrial Research, New Delhi, India.
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Competing interests
The author(s) declared no potential con icts of interest with respect to the research, authorship, and/or publication of this article.

Research involving Human Participants and/or Animals
This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent
Formal consent is not required.    TP-1, hTR, hTERT mRNA was observed after P19 treatment. Beta-actin was considered as an internal control,* represents the statistically signi cant (P<0.05) difference between control and P19 treated cells.
Data are representative from one of three similar experiments.