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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Induction of apoptosis, anti-proliferation, tumor-angiogenic suppression anddown-regulation of Dalton’s Ascitic Lymphoma (DAL) inducedtumorigenesis by poly-L-lysine: A mechanistic study

Souvik Debnatha, Avinaba Mukherjeeb, Saumen Karana, Manish Debnathc,Tapan Kumar Chatterjeea,d,⁎

a Division of Pharmacology Research Laboratory, Department of Pharmaceutical Technology, Jadavpur University, Jadavpur 700032, IndiabDepartment of Zoology, Charuchandra College, University of Calcutta, Kolkata 700029, Indiac Department of Organic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, Indiad Department of Pharmaceutical Technology, JIS University, Kolkata 700109, India

A R T I C L E I N F O

Keywords:Poly-L-lysine(PLL)Anti-cancerTumorigenesisApoptosisAnti-proliferativeAngiogenesisSolid-tumorCaspase-3

A B S T R A C T

Purpose: The present study, attempts to validate the molecular mechanism(s) of Poly-L-lysine (PLL) inducedapoptosis, anti-proliferative and anti-tumorigenic properties in in–vitro HUVECs cells and Dalton’s AsciticLymphoma (DAL) and in in-vivo DAL cell bearing BALB/c mice model.Materials and methods: The cell proliferation assay and morphological assay was carried out using the MTT assayand Giemsa staining method. The antitumor activity of PLL was evaluated in BALB/c mice at 20 and 40mg/kg/b.w doses for 21 days for DAL solid tumor model. Several tumor evaluation endpoints, hematological andbiochemical parameters were estimated. Additionally, the tumor apoptosis, anti-proliferative and anti-tumorangiogenesis effects were assessed using western blots and immunohistochemistry.Results: PLL significantly decreased cell proliferation in in-vitro HUVECs and DAL cells without significant effectson normal cell growth. PLL also induced alteration in cellular morphology in DAL cells. Therafter, in the BALB/cmouse model, PLL had noticeable inhibition in DAL-induced tumorigenesis. This inhibition was evident throughreduced solid tumor volume and weight versus the control group. However, PLL promoted tumor apoptosis andsuppressed cell-proliferation and tumor-angiogenesis. PLL also increased hematological markers significantlycompared to 5-flurouracil (5-FU). The amount of TdT in the nuclei of DAL cells in mice treated with PLL wassignificantly increased while in contrast decreases of anti-apoptotic protein Bcl-2 expression were observed. PLLalso significantly upregulated the pro-apoptotic protein Bax and activated caspase-3. Measurable decreases ofcyclin-D1 were observed through PLL treatments, an indicator of cell-cycle arrest. These studies also indicatePLL’s induction and anti-proliferative effects through suppression of the c-Myc and Ki-67 proliferation-indices.Additionally, PLL inhibited tumor-angiogenesis through suppression of VEGF and CD34 protein expression levelsand reduction ofmicrovesseldensityversus similar parameters in tumors from control mice.Conclusion: The present study offers opportunities and hopes for possible anti-tumortherapies with PLL in thenear future and warrants further formulation developments.

1. Introduction

Cancer is a major leading public health problem worldwide and thesecond leading cause of death after cardiovascular diseases [1]. Severalapproaches, including surgery, chemotherapy and radiation therapy,are available for the treatment of cancer, but they often lack targetspecificity that compromises therapeutic benefits [2]. Thus the

imperatives for novel drugs for cancer therapies with improved speci-ficity andsafetyishighlydesirable.

Apoptosis or programmed cell death is an essential cellular processfor cell rejuvenation and maintenance of proliferation, growth inhibi-tion and death [3,4]. Apoptosis may be triggered by activation of themitochondrial (intrinsic) pathway, resulting from activation of cascadefamily proteases [5]. In this process, Bcl-2 proteins are considered

https://doi.org/10.1016/j.biopha.2018.03.076Received 1 December 2017; Received in revised form 12 March 2018; Accepted 13 March 2018

⁎ Corresponding author at: Dean, Dept. of Pharmaceutical Science and Technology, JIS University, Kolkata. Former Professor, Division of Pharmacology Director, Clinical ResearchCentre (CRC), Jadavpur University, Kolkata-32, India.

E-mail addresses: [email protected] (S. Debnath), [email protected] (A. Mukherjee), [email protected] (S. Karan),[email protected] (M. Debnath), [email protected] (T.K. Chatterjee).

Biomedicine & Pharmacotherapy 102 (2018) 1064–1076

0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

T

important regulators. These onco proteins are classified into twogroups: (i) anti-apoptotic or apoptosis inhibitors and (ii) pro-apoptoticor apoptosis accelerators. The up-regulation of the pro-apoptotic pro-tein Bax and down-regulation of anti-apoptotic protein Bcl-2 is thecrucial part of the apoptotic process [6,7]. The Caspase-3 down streamprotein, a major effector, when activated triggers the apoptotic process[8,9].

Angiogenesis is a determinant of growth and proliferation of solidtumors leading to the formation of new blood vessels via endothelialcells from existing micro-vessels [10,11]. Endothelial cell survival,proliferation, migration, organization and remodeling into the capil-lary-like structure are dependent on angiogenesis [12]. Sustenance oftumors depends on induced angiogenesis via secreted angiogenic fac-tors from tumor cells. The vascular endothelial growth factor (VEGF) isthe most potent mediator of angiogenesis [13], that stimulates tumorprogression, invasion and metastasis [14]. Inhibiting tumor angiogen-esis by suppression of VEGF protein expression can possibly halt tumorgrowth and decrease their metastatic potential [15].

Ki-67, a non-histone nuclear protein, is intimately linked to cellproliferation and cell cycling. It is expressed in proliferating cells duringmid G1 phase, with levels increasing through S and G2 phases. It arrestscell at the M phase proliferating cycle. Recent studies indicate the up-regulation of Ki-67 along with the indexes of Bcl-2/Bax may be corre-lated with various cancer prognosis [16,17]. c-Myc, a transcriptionregulatory oncoprotein, also controls cell cycle, cell apoptosis andprotein synthesis [18]. c-Myc the transcription regulatory oncoprotein,activation and tumor suppressor gene inactivation, leads to DNA repair

system alterations and apoptosis regulation. Such activation-inactiva-tion in the phenomenon in proliferating cells could make c-Mycan in-hibition target for cancer therapy [19].

Poly-L-lysine, (PLL) has some unique biological properties includingan early report on its limited activity in murine tumors. PLL is cationicand aids in the cell membrane permeation of actives in cancer cells[20]. PLL exhibits cytotoxic effects in HeLa and L1210 murine leukemiacells [21,22].

Our previous study on PLL confirms that it inhibits the growth oftumor cells through reduction of Bcl-2 and CD31 expressions and in-creasing Bax, p53 protein expressions and cell-cycle stasis in in-vivoEhrlich ascites carcinoma (EAC) and Sarcoma-180 tumor models. PLLalso reduces peritoneal angiogenesis and microvessel density in EACmice.

However, the precise molecular and associated anti-tumorigenicmechanism(s) of PLL remains unknown. In an effort to elucidate PLL’smechanistic anti-tumorigenicity [23] this study was designed to de-termine the cell proliferation inhibitory effect of PLL on Human um-bilical vein endothelial cells (HUVECs) and Dalton's Ascitic Lymphoma(DAL) in-vitro. To substantiate the possible positive effects in the in vitromodels, anti-tumor activity, apoptotic, anti-proliferative and angio-genic suppressive effects of PLL against Dalton’s lymphoma solid tumormodel were studied in BALB/c mice as an in vivo model.

Fig. 1. Chemical structure of poly-L-lysine (PLL) (A). PLL inhibits cell proliferation in HUVECs and DAL cells. HUVECs (B) and DAL (C) cells were exposed to variousconcentrations of PLL at various dosed for 24, 48 and 72 h and cell proliferation assays were performed. Data are exposed as mean ± SEM, n=3. HUVECs: HumanUmbilical Vascular Endothelial cells; DAL: Dalton’s Ascitic Lymphoma cell.

S. Debnath et al. Biomedicine & Pharmacotherapy 102 (2018) 1064–1076

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2. Materials & methods

2.1. Chemicals & reagents

PLL hydrobromide (Mrs 30,000–70,000) and 3 – (4,5-Dimethytthiazol-2-yl) −2, 5 –diphenyltetrazolium bromide (MTT)were obtained from Sigma-Aldrich (USA). Secondary anti-bodies Bax,Bcl-2, cleaved caspase-3, c-Myc, and GAPDH were purchased fromSanta Cruz, CAUSA. Dulbecco's modified Eagle medium (DMEM),Roswell Park Memorial Institute medium-1640 (RPMI-1640), Penicillin,streptomycin and neomycin (PSN), fetal bovine serum (FBS), trypsinand ethylene diamine tetra-acetic acid (EDTA) obtained from HiMedia(India). Horseradish peroxide (HPR) and VEGF were purchased from R&D systems (USA). A primary antibody for immuno histochemistrystudy CD34, Ki-67, TdT and cyclin-D1 were obtained from Santa CruzBiotechnol. Inc., CA USA. All water-soluble compounds were dissolvedin Dulbecco's Modified Eagle's medium (DMEM), whereas in solublechemicals were dissolved into dimethyl sulfoxide (DMSO) (Sigma,USA), the final concentration of DMSO in each sample was less than0.1%. All solutions were passed through a 0.22 μm fitter (GVMP 01230,Millipore) and stored at 4 °C until use. The other chemicals used were ofanalytical grade.

2.2. Preparation of the drug solution and selection of drug dose by acutetoxicity study

The solution of PLL was prepared by dissolving dry lyophilized PLLhydrobromide (Fig. 1A) in sterile phosphate buffered saline (PBS, pH:7.4). The solution was kept at 4 °C to maintain stability for extendeduse.

For acute toxicity study, mice (n=6) were reared under suitablecondition (room temperatures – 23 ± 2 °C, humidity – 50 ± 5% and12 h light/dark cycle) and were given food and water ad libitum. Theywere given PLL at 20mg/kg bw, 40mg/kg bw and 60mg/kg bw underthe suitable condition. Thereafter, their behaviour was observed after24 h of PLL exposure.

2.3. Cell line and cell culture

In-vitro HUVEC cell line was obtained from National Centre for CellScience (NCCS), Pune, India and Dalton's Ascitic Lymphoma (DAL)cancer cell was collected from Chittaranjan National Cancer ResearchInstitute (CNCRI), Kolkata, India.

HUVEC cells being an endothelial in origin shows highly pro-liferative activity. To show anti-proliferative efficacy of any drug mo-lecules, this cell is being choosen. Here, HUVEC were used from pas-sages 4 to 10. HUVECs were maintained in endothelial cell medium(ECM) containing endothelial cell growth supplement (ECGS).

The tumor Dalton’s lymphoma(DAL) was originated in the thymusgland of a DBA/2 mouse model. DAL is a transplantable T-cell type non-Hodgkin lymphoma that begins in the lymphatic systems [24]. In micemodel, DAL induces solid malignant tumor because it is a spontaneous,undifferentiated cell line, hyperdiploid in nature having high trans-plantable capability [25,26].

The in-vitro HUVAC, DALcells were maintained in DMEM. Besidethis, peripheral blood mononuclear cells (PBMC) was isolated from ahealthy human donor and maintained in RPMI-1640 medium. All thecells were incubated with 5% fetal bovine serum (FBS) and 100 μg/mleach of penicillin and streptomycin, cells were incubated at 37 °C in ahumid atmosphere (5% CO2; 95% air). Cells were harvested by briefincubation in 0.02% (w/v) EDTA in PBS. The cells were maintainedroutinely in subcultures in tissue culture flasks. For in-vivo experiments,the DAL cell was maintained in the peritoneal cavity of mice by in-jecting 0.1 ml at fluid cell every 7 days. Tumor cell counts were done ina Neubauer hemocytometer using the trypan blue dye exclusionmethod. Cell viability was always found to be 95% or more [27]. Tumor

cell suspensions were prepared in phosphate buffered saline (PBS).

2.4. Animals

Eight to ten per cage of healthy female BALB/c mice weighing about20 gm were kept for at least 14 days in environmentally controlledroom temperatures (23 ± 2 °C), humidity (50 ± 5%) and light (12 hlight/ dark cycle) and were given food and water ad libitum. All ex-periments were conducted as per guidelines cleared by the AnimalEthics Committee of the Department of the Pharmaceutical Technologyof Jadavpur University, India (Registration number: 147/1999/CPCSEA).

2.5. HUVECs and DAL proliferation inhibition assay

The effect of PLL on growth inhibition was measured by MTT assay.Briefly, HUVECs and DAL cells at a density of 5×104 cells/well intriplicate were plated onto 96-well flat bottom culture plates withvarious concentrations of PLL (0.5, 1.0, 5.0, 10.0, 20.0 and 30.0 μM)test compound. All cultures were incubated for 24, 48 and 72 h at 37 °Cin a humidified incubator with 5% CO2 in the atmosphere. HUVECswere incubated in serum-starved ECM for 24 h and co-treated withVEGF (20 μg/ml) in the absence or presence of test compound. Afterincubation, MTT reagent was added to a final concentration of 0.5mg/ml and further incubated for 4 h. The superintendent was discarded andprecipitated formation salt was dissolved in DMSO and read at 490 nmwith an ELISA microplate reader (Bio-rad, USA). The inhibition ratio(%) was calculated by the equation and expressed as the average ofthree parallel experiments. Beside this PBMC cells were also examinedby MTT assay after being exposed with the PLL of same doses.

= ⎛⎝

− − ⎞⎠

×A blank treated A blankA

1% 1 100%

2.6. Study on change in morphology of DAL cells by Giemsa stainingmethod

For this study, 18 animals were divided into 3 groups (6 animals pergroup), e.g., group I–III respectively. DAL Ascites fluid was drawn outfrom DAL tumor bearing mouse at the log phase (day 7–8 of tumorbearing) of the tumor cells. Each animal was inoculated with 0.1 ml oftumor cell suspension, prepared in phosphate buffer solution (PBS)containing 2×106 cells/ml. Group-I was considered as a control. After24 h of DAL cell transplantation, group-II and III of the experimentalanimals received PLL at doses of 20 and 40mg/kg b.w, i.p. respectively,for 14 consecutive days. After complete ion of drug induction, the an-imals were fasted for a period of 18 h, followed by sacrifice by cervicaldislocation. 2ml of PBS was injected (i.p.), and a small incision wasmade in the abdominal region to collect the tumor cells along withascites fluid. Then DAL cells along with the ascites fluid were harvestedinto 15ml. centrifuge tubes and centrifuged at 3000 rpm for 10min at4 °C. Then, DAL cells from the control and test drug treated groups weresmeared on clean glass slides, air-dried, and fixed in a solution of me-thanol/acetic acid (3:1). The slides were hydrated with PBS & thenstained with 0.1% Giemsa solution and observed under compound lightmicroscope (Eclipse TS100, Nikon, Japan).

2.7. Induction of DAL solid tumor

In the DAL solid tumor model, 24 animals were assigned to 4 groups(6 animals per group), e.g., group I to IV, respectively. Ascites fluid wasdrawn out from DAL tumor-bearing mouse at the log phase (day 7–8 oftumor-bearing) of the tumor cells.

Each group was given subcutaneous inoculation (s.c.) of 0.1ml DALcell suspension containing 2× 106 cells/ml. Group-I served as thecontrol, followed by treatments groups-II and III, receiving doses of (20


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and 40mg/kg of b.w) of PLL (i.p), respectively and the Group-IV re-ceived 5-FU, as reference drug (20mg/kg i.p.). Treatment was started10 days after tumor inoculations and was continued for 21 days, similarto the solid tumor model [28]. The tumor volume was determined bydirect measurement with verniercalipers every 2 days after the tumorimplantation and the tumor inhibition was calculated [29,30]. On day22, the animals were sacrificed by cervical dislocation and the tumorswere removed for evaluation of various antitumor activities.

2.8. Tumor volume

The ratio of the developing tumors was measured usingVerniercalipers at 2-day intervals for 21 days and the tumor volumewas calculated using the formula:

V= π/6× (D1)× (D2)2 (2)

Where D1 is the longer diameter and D2 is the shorter diameter.

2.9. Tumor weight

At the end of 21 days, all tumors (2 discs/animals) was punched out,weighed immediately, and the average weights were calculated. Alltumors were excised and divided into two portions. The first portionwas used for identification of protein expression levels, and the secondwas used for histopathological and immuno histochemical measure-ments. The percentage inhibition was calculated by the formula:

%Inhibition=1-B/A×100

Where A is the average weight of the control group and B is the averagetumor weight of the treated group.

2.10. Estimation of hematological and serum biochemical parameters

After 21 days from the outset of the experiment, animals were sa-crificed by cervical dislocation and blood samples were collected fromthe heart using heparinized syringes for hematological and serum bio-chemical parameters.

2.11. Percentage increase in life span (%ILS)

After sacrifice, 3 mice from each group were observed for meansurvival time (MST). The effect of PLL on the percentage increase inlifespan (%ILS) was calculated on the basis of mortality of the experi-mental mice. Mortality was monitored by recording (% ILS) and (MST)as per the following formula:

=∑

Mean Survival timesurvival time (days) of each mouse in a group

Total number of mice

= ×%ILS MST of treated animalsMST of control animals

100-100

Here, the time is denoted byanumber of days.

2.12. H&E staining of sarcoma-180 tumor section

On day 21, the BALB/c mice were sacrificed, tumors that developedat the site of injection were excised and fixed in 10% formaldehyde andembedded in paraffin and 5 μm sections were stained with hematoxylinand eosin (H&E). The slides were examined for histopathologicalchanges such as a necrosis, mitotic figures, and inflammatory reactionsusing light microscopy. 3 Animals per group were used for histologicaland immunohistochemical analysis.

2.13. Immuno histochemistry analysis

Thick tumor sections (∼5 μm) were used for immunohistochemicalanalysis of Ki-67, TdT, cyclin-D1 and CD34 proteins. Briefly, the sec-tions were hydrated in 1 X PBS for 5min. Antigen retrieval was per-formed by incubating the sections in 10mM sodium citrate buffer (pH6.0) at 80 °C for 10min. The sections were cooled to room temperaturefor 20min. Following a 5-min wash with 1 X PBS, the endogenousperoxides were blocked by 1% hydrogen peroxide in PBS for 5min. Thesections were washed as before and blocked for 1 h. in PBS containing1.5% normal serum. The slides were incubated overnight with primaryantibodies respectively against Ki-67, TdT, cyclin-D1 and CD34 at 4 °Cin a humidified chamber. After washing with PBS, the sections wereincubated with horseradish peroxides (HRP) – conjugated secondaryantibodies at 1:100 dilutions for 30min. at 37 °C. The immune reactionswere visualized by immersing the slides in 3,3′-diaminobenzidine tet-rahydrochloride reagent. The sections were counterstained with he-matoxylin. Negative control sections were processed simultaneouslywith the omission of the primary antibodies. All sections were dehy-drated, mounted viewed under a light microscope (Eclipse TS100,Nikon, Japan) and photographed (10X).

2.14. Western blot analysis

DAL solid tumor tissues were harvested from control and drug-treated mice. At the end of the treatment period, tumor tissues werehomogenized in RIPA buffer kit (3D Bioscience, U.S.A.) is the presenceof protease inhibitors. Cell lysates were collected and the total proteincontents estimated by the Lowry method. The protein (30 μg) contentsfrom the cell lysates were separated by 10% SDS–PAGE and transferredto a nitrocellulose membrane. The membranes were blocked, washedand the respective membranes were probed using antibodies for Bax,Bcl-2, cleaved caspase-3, c-Myc, VEGF and GAPDH (as loading control)overnight at room temperature. GAPDH was taken as the housekeepinggene. The blots were washed and immunoreactive bands were in-cubated with a 1:2000 dilution of HRP (horseradish peroxidase) con-jugated secondary antibody for 2 h at room temperature. Binding sig-nals were visualized with TMB (3,3′,5,5′ Tetramethylbenzidine)substrate. Relative band intensities were determined with ImageJsoftware.

2.15. Statistical analysis

All the results values were expressed asamean ± standard error ofthe mean (SEM). Experimental results were analyzed by student’s t-testand One-way analysis of variance (ANOVA), followed by Kruskal WallisTest using SPSS statistical software of 20.0 version. The survival ratewas analyzed by Kaplan-Meier method. *P < 0.05 and ***P < 0.001were considered to be statistically significant when compared withcontrol.

3. Results

3.1. Poly-L-lysine inhibited the proliferation of HUVECs and DAL cell

To examine the effect of PLL on the proliferation of HUVECs andDAL, cells were treated with different concentrations of PLL (0, 0.5, 1.0,5.0, 10.0, 20.0 and 30.0 μg/ml) for 24, 48 and 72 h and their pro-liferation were measured. After 72 h of exposure, 0.5 μg/ml PLL did notimpair the effective proliferation, however, aconcentration of1.0–30.0 μg/ml it potently diminished the cell proliferation 82.4–10.7%respectively, as compared to control (Fig. 1B). However, when PLL wasapplied against PBMC, showed no cytotoxicity indicating its targetspecificity.


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3.2. Changes in the morphology of DAL cells after PLL treatment

The inhibitory effect of PLL on DAL cells of the mice model wasobserved by Giemsa staining. Group II and III corresponds to Giemsastaining that demonstrates blebbing of plasma membrane (marked byarrow), shown, whereas, the formation of apoptotic bodies could alsobe observed in (Fig. 2). In general, the control (DAL cells) cells (GroupI) have good morphology with intact plasma membrane whereas, incase of the test drug, the apoptotic bodies and the nuclear condensationis evident (marked by arrow in Fig. 2). In the PLL drug treated group forthe Group II that receives 20mg/kg b.w. of PLL, chromatin condensa-tion, blebbing of plasma membrane, irregularity in cell morphology, isobserved in (marked by arrow in Fig. 2). As the dose of PLL is escalatedto twice the former, i.e., 40mg/kg b.w. in case if Group IIImice, ex-hibited in both the phenomenon of cellular blebbing, followed by cellshrinkage, cytoplasmic irregularity, nuclear fragmentation irregularshape and formation of apoptotic body as a dose dependent manner.

3.3. Selection of dosimetry of PLL for in-vivo experiments

As per earlier report of Arnold et al. [20], PLL solution was

administered by intraperitoneal injections (i.p.) of 20mg/kg b.w. inmice. Therefore, in the present study, the same dose (20mg/kg b.w.) ofPLL was taken. On the other hand, 5-FU was used as a standard drug at40mg/kg b.w dose [31]. So, to compare the efficacy of our test drug,PLL with the standard drug, 5-FU the same dosimetry (40mg/kg b.w.)was taken along with 20mg/kg b.w. However, preliminarily, acutetoxicity test was done in mice after being intoxicated with 20mg/kgb.w., 40mg/kg b.w. and 60mg/kg b.w. of PLL for 24 h. Results in-dicated that there is no behavioural changes in mice having 20 and40mg/kg b.w. of PLL. But, partial behavioural changes and rapidmovement were observed in mice having 60mg/kg b.w. of PLL.Therefore, for further studies, PLL was taken at the dosimetry of 20 and40mg/kg b.w. and administered by intraperitoneal injections (i.p.) inDAL induced tumorigenic mice.

3.4. Effect of PLL on hematological parameters

Haematological parameters of PLL treated and untreated (21 days)tumor-bearing mice were evaluated (Table 1). Haemoglobin and RBClevel that goes down generally during the progression of the tumor wasfound to improve in mice treated with PLL, PLL treatment reduced WBC

Fig. 2. Change in morphology of the DAL cells by Giemsa stain control, PLL 20mg/kg and PLL 40mg/kg b.w. by giemsa staining of Fig. 4 show cellular blebbing andapoptotic body respectively and show nuclear fragmentation.

Table 1Effect of PLL on hematological parameters in DAL bearing mice.

Poly-L-lysine(20 mg/kg b.w) Poly -L-lysine(40 mg/kg b.w) Std. Drug 20mg/kg b.w DAL Control (2× 106 cells/mouse) Normal Mice

Hemoglobin (gm %) 8.45 ± 0.01* 8.75 ± 0.02* 8.88 ± 0.02* 6.44 ± 0.19* 12.4 ± 0.14Erythrocyte(RBC)(million/mm) 6.96 ± 0.02* 7.31 ± 0.02* 6.7 ± 0.02* 4.38 ± 0.02* 9.59 ± 0.03Leucocytes (WBC) 12.37 ± 0.03* 9.79 ± 0.02* 8.25 ± 0.02* 18.14 ± 0.03* 13.5 ± 0.03Neutrophil (%) 34.17 ± 0.02* 30.15 ± 0.03* 41.20 ± 0.02* 72.18 ± 0.06* 30.07 ± 0.06Lymphocyte (%) 44.56 ± 0.11* 53.83 ± 0.02* 61.05 ± 0.04* 34.06 ± 0.05* 68.32 ± 0.25Monocyte (%) 1.72 ± 0.02* 2.31 ± 0.03* 1.75 ± 0.02* 1.18 ± 0.03* 2.16 ± 0.03

Each point represents the mean ± SEM. (n=6 mice per group).* p < 0.05 statistically significant when compared with normal saline group and DAL control group.

Table 2Effect of PLL on serum biochemical in DAL bearing mice.

Poly-L-lysine(20 mg/kg b.w) Poly-L-lysine(40mg/kg b.w) Std. Drug 20mg/kg b.w DAL Control (2× 106 cells/mouse) Normal Mice

Bilirubin Total & Direct (mg/dl) 0.31 ± 0.01* 0.26 ± 0.02* 0.26 ± 0.01* 0.37 ± 0.01* 0.42 ± 0.02Conjugated bilirubin(mg/dl) 0.16 ± 0.04* 0.12 ± 0.03* 0.13 ± 0.04* 0.14 ± 0.04* 0.21 ± 0.02Unconjugated bilirubin(mg/dl) 0.15 ± 0.04* 0.14 ± 0.04* 0.13 ± 0.03* 0.23 ± 0.04* 0.21 ± 0.01Serum Protein (Total)(g/dl) 6.28 ± 0.08* 6.62 ± 0.08* 6.74 ± 0.11* 2.34 ± 0.11* 6.82 ± 0.08Albumin(g/dl) 2.22 ± 0.77* 2.92 ± 0.29* 2.82 ± 0.37* 1.2 ± 0.07* 2.92 ± 0.44Globulin(g/dl) 4.06 ± 0.7* 3.7 ± 0.37* 3.88 ± 0.35* 1.14 ± 0.05* 3.9 ± 0.37AST(SGOT) 55.38 ± 0.07* 35.1 ± 0.03* 29.12 ± 0.02* 79.05 ± 0.05* 37.14 ± 0.03ALT(SGPT) 46.18 ± 0.02* 32.33 ± 0.04* 30.08 ± 0.04* 64.35 ± 0.03* 29.31 ± 0.03Serum Alkaline Phosphatase 96.25 ± 0.03* 79.12 ± 0.02* 73.94 ± 0.03* 123.26 ± 0.03* 77.25 ± 0.01Creatinine (mg/dl) 0.76 ± 0.01* 0.8 ± 0.02* 0.56 ± 0.01* 0.62 ± 0.02* 0.82 ± 0.02

Each point represents the mean ± SEM. (n=6mice per group). *p < 0.05 statistically significant when compared with normal saline group and DAL control group.* P < 0.05 v/s DAL control group.


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count compared with the DAL control. In the differential count of WBC,lymphocytes and monocytes decreased and neutrophils increased in theDAL control group when compared with healthy controls. PLL treatedat different doses as above significantly changes the relevant para-meters approximately, to the normal values (Table 1).

3.5. Effect of PLL on biochemical parameters

Table 2 demonstrated that the biochemical parameters like SGOT,SGPT, SALPand bilirubin significantly decreased in a dose-dependentmanner (20 and 40mg/kg b.w.) as compared to DAL control group.Results also show that total protein content increased dose-depen-dently.

3.6. PLL inhibits DAL solid tumor growth in mice

The average tumor volume in DAL control mice progressively in-creased with time upto 68.99 ± 1.514mm3 from 10.58 ± 5.86mm3

(at day 1st) for 21 days post-tumorimplantation study. In PLL test drugtreated groups the percentage oftumor volume was observed in respectto control group are 32.96 ± 0.07 for 20mg/kg b.w and 59.18 ± 0.06for 40mg/kg b.w. which indicats tumer volume decrease dose-depen-dently (Fig. 3A, B and D). Besides, PLL treatment significantly reducedtumor weight compared to DAL bearing control group (***P < 0.001)mice (Fig. 3C). The body weight changes were significantly higher inPLL treated groups compared to control indicating the effect of PLL inpreventing the tumor growth (Fig. 3D).

Fig. 3. Effect of PLL treatment on solid DAL tumor. 2× 106 cells/mouse were injected s.c into 5–6 week old BALB/c mice. (B) After solid tumor grow to ̴100mm3, themice were i.p treated with PLL [20mg/kg b.w, 40mg/kg b.w and standard drug 5FU (20mg/kg b.w)] for 21 days. (A) and (B) PLL inhibited the growth of solidtumors. (C) Change the body weight between drug treated and control group. (D) Tumor volume percentage and solid tumors lump in the PLL treated mice significantby smaller than those in the control mice. Data are reported as the mean ± SEM of three different observations (6 animals per treated group). ***p < 0.001 ascompared to control group.

Table 3Effect of PLLonTumor weight, Mean Survival Time (MST), Increased Life Span (ILS) and Bodyweight.

Poly-L-lysine (20mg/kg b.w) Poly -L-lysine (40mg/kg b.w) Std. Drug (20mg/kg b.w) DAL Control (2× 106 cells/mouse)

Tumor Weight (gm) 7.15 ± 0.04*** 4.16 ± 0.05*** 3.12 ± 0.04*** 19.28 ± 0.16% of Tumor Volume 81.25 ± 0.07*** 68.35 ± 0.06*** 62.58 ± 0.07*** 0 ± 0Tumor growth Inhibition (%) 24.35 ± 0.05*** 36.97 ± 0.08*** 46.90 ± 0.12*** 0 ± 0MST (days) 77.16 ± 0.15*** 84.38 ± 0.28*** 59.50 ± 0.34*** 25.41 ± 0.27***

% ILS 39.59 ± 0.08*** 57.89 ± 0.82*** 49.58 ± 0.27*** 0 ± 0Body weight (gm) 22.64 ± 0.54*** 18.52 ± 0.71*** 17.28 ± 0.13*** 38.02 ± 0.56***

Each point represents the mean ± SEM. (n= 6 mice per group).*** p < 0.001 statistically significant when compared with DAL control group.


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3.7. Effect of PLL treatment on the survival of tumor-bearing mice

As shown in Table 3 and Kaplan-Meier survival curve in Fig. 4, PLLtreatment significantly increased the survival of DAL bearing micecompared to the DAL bearing controlled mice (***P < 0.001). The %

ILS in PLL at 20 and 40mg/kg was found to be in a dose-dependentmanner (Fig. 4).

Fig. 4. Kaplan-Meier survival curve for PLL treated and untreated groups.

Fig. 5. Effect of PLL treatment on solid DAL tumor. (A) In H&E staining of solid DAL tumor, microphotograph showing sheets of tumor cells. Mitotic figures andhigher chromatophilicor cells at variable shape representing cell proliferation surrounding areas at necrosis in DAL control mice. Microphotograph is showinga-ninflammatory reaction after treatment 20mg/kg b.w and tumor cells lying freely in dilated space and sheets of malignant cells with focal necrosis and apoptosis.Microphotograph showing smooth margins of the tumor without infiltration in surrounding tissues treated with PLL 40mg/kg b.w. There is also showing reducedmitotic figures and apoptosis with PLL 40mg/kg b.w treatment. (H&E 400×). (B) The quote of the percentage of necrotic area in sections of tumors harvested on d 22post-treatment. In each section per tumor four random areas were counted and three tumors were harvested in each treatment group. The quota of the percentage ofnecrosis was determined using Image J software. Mean ± SEM, n=3.


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3.8. Effect of PLL on histopathological changes of solid DAL tumor

Histological examination was done through H&E staining of tumorsections. Our results indicate that tumor tissue derived from DAL con-trol mice has a disordered arrangement of cancer cells and a high celldensity. The cells were voided or round and showed a high nucleuswhich was thickly stained with obvious signs of heteromorphism andhyperplasia. Also,it showed intact anaplastic cancer cells with areas ofnecrosis and focal hemorrhage reflecting the viability and aggressive-ness of the tumor invading cellular infiltration, cords of malignant cellsand large tumor cells in between muscle fibers, high mitotic activity inDAL bearing mice. As shown in Fig. 5 and Table 4 the number of tumorcells in the PLL treatment groups decreased marked by cells werepolygonal and light stained. Besides, tumor cell nuclei shapes were ir-regular and the surface of the nucleic membrane was rough. The nu-cleus was broken, less tumor invasion and cellular in filtrates, moderatemitotic activity and markedly increased apoptotic bodies. The mice ofthe PLL treated groups exhibited smaller necrotic areas and reduce thenumber of mitotic figures.

3.9. PLL inhibits tumor-induced neovascularization in DAL solid tumor

CD34 is one of the most commonly used endothelial cell markersused to highlight tumor blood vessels immunohistochemically. It is asurface glycoprotein that highlights perivascular stromal cells. CD34analysis showed numerous large blood vessels in DALcontroltumor. PLLsuppressed CD34 expression in DAL solid tumor (Table 5; Fig. 6C).CD34 has been used as a marker for microvessel density (MVD) esti-mation and MVD values have been compared among the variousgroups. As evident from (Fig. 6D) PLL (20mg and 40mg/kgb.w.) de-creased the % MVD as compared to control mice in a dose-dependentmanner, indicating potent anti-angiogenic activity of the PLL.

In (Fig. 6A and B), further to validate this effect, I had done westernblot study for an angiogenesis marker. VEGF is the key angiogenesisgrowth factor. This result clearly indicates that in the DAL solid tumorPLL treated mice, a significant reduction of VEGF level as the dose-dependent manner was observed when compared to the VEGF level

from DAL control group mice.

3.10. PLL administration suppresses cell proliferation and induces apoptosisin in-vivo DAL solid tumor

There after, studies have been made on PLL mediated suppressionthe DAL induced solid tumor growth was accompanied by inhibition ofcell proliferation and apoptosis induction in tumor tissues. As observedin Fig. 7A, control DAL tumor sections revealed strong nuclear Ki-67expression hinting at expressive cell proliferation. In contrast, PLL ad-ministration resulted in significant decrease of Ki-67 positive cells andproliferation index (Fig. 7B; Table 6).

Consisting of the in-vivo data, PLL administration also elicitedapoptosis is the solid tumors shown by immuno histochemistry (IHC) ofTdT marker (Fig. 8A and B). Following PLL treatment, subcutaneoustumor tissue showed increased cellular injury and a striking stainingpattern of abundant TdT positive cells with focal concentration. HereIHC result indicated that PLL induced tumor cell apoptosis indicated byan increase of the TdT-positive cell numbers.

Together, these results indicated that PLL administration inhibitedsubcutaneous DAL solid tumor growth in-vivo by reducing cell pro-liferation and inducing apoptosis.

3.11. PLL attenuates the expression of cyclin-D1

In the last investigation, PLL has the ability to induce apoptosis byblocking the cell cycle progression at the sub G1 phase [23]. Further-more; mechanism of PLL induced cell cycle arrest by modulation ofcyclin-D1 if any was evaluated by immunohistochemical analysis. Re-sults indicated that the level of cyclin-D1 decreased due to the treat-ment of PLL in a dose-dependent manner (Fig. 9). As cyclin-D1 bindsand activates cyclin-dependent kinase 4 and 6 (CDK4 and CDK6) whichregulate G1/S transition [32]. These results suggested that PLL inducedthe cell cycle arrest through regulating the expression of the cell-cycle-related protein.

3.12. PLL potentiated the anti-tumor and anti-proliferative effects in DALsolid tumor in-vivo model

Apoptosis is regulated by various proteins such as Bax, Bcl-2,Caspase-3 and c-Myc [33–36]. Western blot analysis was performed onproliferation and apoptosis-related proteins (Bax, Bcl-2, cleaved cas-pase-3 and c-Myc) to identify the mechanism by which PLL attenuatesDAL cell growth (Fig. 10A and B). PLL significantly up-regulated theexpression of cleaved caspase-3 in the PLL treated cells compared withthe respective DAL control group as dose-dependent manner(***P < 0.001). PLL suppressed the expression of the anti-apoptoticprotein Bcl-2 and up-regulated that of the pro-apoptotic protein Bax.Our results demonstrated that PLL significantly increased Bax/Bcl-2ratio in comparison with DAL control group (***P < 0.001).

PLL dose-dependently (***P < 0.001) suppressed the protein ex-pression of c-Myc in DAL solid tumors, while it attenuated the expres-sion of c-Myc alone in DAL cells (Silencing of c-Myc promoted theability of PLL to sub G1 accumulation in DAL solid tumor).

4. Discussion

Cancer is an intractable disease. Currently, there is incessant in-terest in the search of efficacious bioactive(s)with lower toxicity for thisterminal disease, worldwide. Apoptosis, or programmed cell death, ischaracterized morphologically by extreme chromatin condensation andformation of apoptotic bodies. It also well recognized that an alterationof the cellular homeostasis occurs in cancer, which disrupts the balancebetween cell proliferation and apoptosis [37,38]. In addition, apoptosisis an orderly type of cell death that progresses through a series ofcascading cell signals [39]. Interestingly, the positive association

Table 4Effect of PLL on the histopathological changes in solid tumor (DAL) bearingMice (n=3).

Group TumorInfiltration atMargin

Inflammatorycells Infiltration atEdge

MitoticIndex (noof MFs in12 HPFs)

Apoptoticindex (no ofAbs in HPFs)

DAL control +++ +++ 12 3PLL (20mg/

kg b.w)++ ++ 5 8

PLL (40mg/kg b.w)

+ + 3 16

Tumor sections from three different tumors in each group, and four randomlyselected areas from each tumor were analyzed (12 HPFs); MF=Mitotic figure;HPF=High power field (×400); Abs=Apoptotic body; (+), mild; (++),moderate; (+++), sever.

Table 5Semiquantitative analysis of immunohistochemicalstaining of CD34 in tumor tissues for molecularmarker (n=3).

Group CD-34

DAL control +++PLL(20mg/kg b.w) ++PLL(40mg/kg b.w) +

+ (Weak Staining); ++ (Moderate staining); +++(Strong Staining).


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between apoptosis and proliferation demonstrated in this study impliesthat apoptosis might reflect not only cell death but it’s proliferation aswell [40].

Results of this study indicates that PLL markedly inhibits pro-liferation of HUVEC and DAL cells sparing the normal cells (PBMC).DAL cells being an as cites tumor shows blebbing in there cellularmembrane, shrinkage in cellular shape and nuclear fragmentation whenbeing intoxicated with PLL as shown by Giemsa staining. Thereafter, forfurther investigation, efficacy of PLL was studied in DAL induced solidtumor in mice model.

The anti-proliferation activity of the PLL against transplantable DALtumors in BALB/c mice was observed and the molecular mechanism(s)of antitumor activity was elucidated. It is important to note that 30 μg/ml of PLL during a 24 h of exposure decreased the HUVEC cell pro-liferation rate to 25%. Further reductions in cell proliferation (up to82.4%) in a time-dependent manner (up to 72 h) were also observedwith PLL at the same concentration.

In the present study, our results showed are duction in tumorweight, volume and tumor size in DAL solid tumor-bearing mice treatedwith PLL and also increased the lifespan of tumor-bearing BALB/c mice.In this study, elevated WBC count, reduced hemoglobin level and RBCcount were observed in DAL control mice and i.p. administration of PLLrestored hemoglobin level and maintained the normal value of RBC &WBC, thus supporting its hematopoietic protecting activity. Also, theliver function was investigated by the biochemical determination atAST, ALT and ALP levels and histopathological examination of liver

tissue in mice given daily i.p. well tolerated doses of 20 and 40mg/kgof body weight. Animals treated with PLL showed no clinical signs ofgross toxicity or changes in behavior. Also, the treatment did not affectthe body weight gain in comparison with the control group. In addition,histopathological examination demonstrated less necrotic area, mitoticfigures count in all treated groups compared to DAL control.

Angiogenesis is integral to tumorigenicity and metastasis. VEGF isthe key determinant in angiogenesis and is over expressed in manycancers [41]. Therefore, VEGF expression loss in a tumor would reducevascular density and permeability dramatically with associated aug-mentation of tumor cell apoptosis [42]. Micro-vessel density (MVD) is aprognostic tool used to assess the degree of tumor angiogenesis bymeasuring CD34 expression [43] compared to PLL treated group wassignificantly decreased the %MVD in the test drug-treated DAL solidtumor model. CD34 bio-molecules are located basally along the lateralendothelial borders and are responsible for adhesion and trans-en-dothelial migrations [44]. In-vivo experiments in this study have shownthat higher CD34 expression(s) were associated with the increasedangiogenic activity, while with inhibition neovascularization was sig-nificantly minimized [45] As shown in (Fig. 6), PLL remarkably reducedthe formation of micro-vessels indicated by the decrease of CD34, amarker of angiogenesis, and down-regulation of VEGF protein in tumortissues. This demonstrates that PLL could act as an inhibitor of angio-genesis to inhibit tumor growth in-vivo.

The abundance of Cyclin-D1 in epithelial cells helps regulate theproduction of VEGF expression. Cyclin-D1 regulates heterotypic signals

Fig. 6. Angio inhibitory activity profile of PLL (A) Immunoblot analysis of VEGF and GAPDH (B) Densitometric analysis showing down-regulation of VEGF proteinexpression. Each value represents the mean ± S.D. of three independent experiments, each performed in triplicate ***p < 0.001 as compared to control group mice.(C) Immuno-staining and optical density of CD34 expression in BALB/c mice (Magnification: 400×) exhibiting the reduction of neovascularization(arrow marked) inthe order as shown. Each bar represents the mean ± SEM, n= 3 tumors. ***p < 0.001 as compared to control group mice.


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to promote vasculogenesis and associated tumor progression. The cy-clin-D1 gene itself is a downstream target of anti-angiogenic growthfactor. VEGF growth factor induces cyclin-D1 expressions. Thus a pos-sibility exists that angiogenic suppressor and an apoptotic moleculeinhibitor would interfere with cyclin-D1 expression leading to induc-tion of G1 and Sub G1 cell cycle arrest [46,47]. Interestingly in thisstudy PLL reduces both VEGF & cyclin-D1 expression and also decreasesthe number of proliferating cells versus control. It is fair to concludefrom these observations that, negative regulators of angiogenesis areinhibited by cyclin-D1 and that cyclin-D1 inhibits VEGF expression.

Remarkably, in the current study, PLL mediated suppression of DALtumor growth in in-vivo mice was accompanied by a marked reductionin cell proliferation and induction of apoptosis, as validated by ki-67and TdT staining of tumor tissue. Percentage of TdT positive cells in-dicated that PLL treatment significantly reduced tumor burden throughincreased numbers of TST positive cells by inducing apoptosis. PLLtumor growth suppression was associated with the reduction of Ki-67, amitosis cell proliferation marker. Ki-67 is a nuclear antigen localized atthe periphery at the chromosome scaffold and nuclear cortex. As per

our results, the relative number of Ki-67 positive cells was substantiallysmaller in tumors from mice treated PLL. These data showed that PLLwas significantly effective in inhibiting Ki-67 expression versus con-trols. The Ki-67 protein is expressed in all phases of the cell cycle exceptG0 and serves as a good marker for proliferation [48,49]. This findingwas in accordance with the previous report which concluded that PLLhas a direct anti-tumor activity through inhibition of DNA synthesisresulting in cell proliferation arrest in the sub-G1 phase [23].

Selective Bcl-2 proteins can regulate cell death including apoptosis.PLL treatment positively correlated with Bcl-2 and VEGF proteins, di-recting apoptotic pathway in cancer. Because elevated Bcl-2 may keepcells in the active cycle and decrease apoptotic level, it can increasetumor cell proliferation. VEGF also facilitates tumor progression byregulating angiogenesis. Our results thus suggest the synergistic effectof PLL, on Bcl-2 and VEGF genes in DAL induced solid tumors. Thisobservation indicates a significant down-regulation of the anti-apop-totic protein Bcl-2 and the up-regulation of the pro-apoptotic proteinBax [28]. When the ratio of pro-apoptotic Bcl-2 family member Bax toanti-apoptotic Bcl-2 family member Bcl-2 increases, pores form in theouter mitochondrial membrane, liberating apopto genic mitochondrialproteins to activate caspase-3 and induce apoptosis and anti-proliferative effect in DAL solid tumor-bearing BALB/c mice. Thesefindings are consistent with previous studies using PLL which showedBcl-2 down regulation, Bax activation in mouse S-180 cancer cell lines[23].

The c-Myc is a multifunctional oncogene involved in cell growth,cellular proliferation, differentiation, apoptosis, tumorigenesis and so isfrequently over expressed in a various type of cancer cells [50]. One ofthe key biological functions of c-Myc is to promote cell-cycle progres-sion in several cancers. After serum stimulation, c-Myc is induced at

Fig. 7. PLL suppressestumor growth in the BALB/c mouse model. (A) PLL administration reduced Ki-67 positive cell populations in DAL solid tumor compared withthe control group. (B) The average percentage of Ki-67 positive cells in tumors arising from PLL treated groups was significantly lower than those in the controlgroup. Each bar represents the mean ± SEM, n=3 tumors. ***p < 0.001 as compared to the control group of mice.

Table 6Ki-67 labelling index (LI) determination.

Group Ki-67 index (%)

DAL control 31.0 ± 5.7PLL 20mg/kg b.w 19.8 ± 6.1***

PLL 40mg/kg b.w 8.5 ± 2.1***

Statistical significant changes as a compound with tumorcontrol group (***p < 0.001). Each point represents themean ± SEM (n=3 mice per group).


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protein levels and the cells enter the G1 phase of the cell-cycle to pro-mote biological process including proliferation or apoptosis [51]. In-terestingly, PLL inhibited the expression of c-Myc inhibition in PLL-induced apoptosis and sub-G1 arrest in the DAL solid tumor model.Also, it is noteworthy that, PLL reduced protein levels of c-Myc by about25% while the protein level c-Myc was also most disappeared by PLL,implying that PLL works on c-Myc rather at the post-transcriptionallevel, through further study is required in the near future.

These suggested that PLL have a direct relationship with tumorcells. Our investigation in-vivo showed that PLL could be a more ef-fective anti-cancer drug, although this needs further intense validation.

Collectively, the findings in the current study demonstrated that thePLL impairs the in-vitro endothelial (HUVEC cell) processes by in-hibiting cell proliferation effect. In summary, all the experimentalfindings are strongly corroborate that PLL showed significant anti-tumor effects through activating immune responses to inhibit tumor cellproliferation and angiogenesis and to induce tumor cell apoptosis in-vivo BALB/c mice model. PLL promoted apoptosis through caspase-3dependent signaling pathway, and inhibited cell proliferation possiblythrough inhibiting Ki-67 positive cells, elevated number of TdT-positivecells and reduce the CD34 protein expression level. Additionally, PLL-induced inhibition of VEGF, c-Myc, cyclin-D1 in tumor tissue could

Fig. 8. PLL induced modulation in TdT expression (A) Immunohistochemical staining of solid DAL tumor section showing the higher amount of TdT in the nuclei ofDAL tumor cells in mice treated with PLL (400×) than the control mice. (B) Apoptotic index, where the average percentage of TdT positive cells in PLL treated groupswere significantly higher than those in control group. Each bar represents the mean ± SEM, n=3 tumors. ***p < 0.001 as compared to the control group of mice.

Fig. 9. PLL suppressestumor growth in the BALB/c mouse model. Cyclin-D1 expressions in solid tumors from mice treated with or without PLL were assessed by IHCstaining, respectively (magnification 400×). Three mice were included for each group and results are representative of three experiments.


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possibly lead to suppression of proliferation and angiogenesis. It is nowobvious from what has been already discussed that PLL likely has asynergistic dual mechanism of action, through its VEGF mediated anti-angiogenesis and caspase-3 mediated apoptosis and therefore is a po-tential powerful chemotherapeutic alternative. Based on these findings,we proposed a schematic presentation of the possible mechanism be-hind the anti-cancer activity of PLL in DAL cells (Fig.11). The importantobservations from this study provide a strong rationale for the possi-bility and considerations for using PLL in cancer therapies.

Conflict of interest

The authors declare that there are no conflicts of interests.

Acknowledgments

University of Grant Commission, New Delhi, India, sanctioned toSouvik Debnath through UGC-BSR Research Fellowship in Science forMeritorious student’s fellowship scheme.

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Fig. 11. Schematic diagram of anti-cancer activity of PLL on in-vitro HUVECs and in-vivo DAL tumor cell.


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