NF-κΒ activator 1

Cathepsin B mediated scramblase activation triggers cytotoxicity and cell cycle arrest by andrographolide to overcome cellular resistance in cisplatin resistant human hepatocellular carcinoma HepG2 cells

Kaustav Dutta Chowdhurya, Avik Sarkarb, Sujan Chatterjeec, Debajyoti Patrac, Dipanwita Senguptad, Soumi Banerjeea, Pratip Chakrabortye, Gobinda Chandra Sadhukhanf*

Highlights:

• 40μM andrographolide after 48hr reduces 50% HepG2CR cell survivability
• Andrographolide significantly up-regulates PKA/PP2A/IKK axis
• Event initiates cathepsinB-tAIF-scramblase mediated ‘eat me’ signal in HepG2CR
• Cyclin A and B downregulation and increase in pTyr15CDK1 result subG1 phase arrest
• No additive/synergistic andrographolide effects with cisplatin against resistance

Abstract

Andrographolide regimen in single or in combination with anticancer drugs is a promising new strategy to reverse chemoresistance in heaptocellular carcinoma. Apoptosis inducing factor (AIF) may regulate a complementary, cooperative or redundant pathway, along with caspase cascades. Despite these findings, mechanisms underlying caspase-dependent and-independent signaling pathways in andrographolide -induced apoptosis in cisplatin-resistant human hepatocellular carcinoma cell line (HepG2CR) remain unclear. Andrographolide treatment effectively reduced NF-κβ nuclear localization by modulating protein kinase A- protein phosphatase 2A- Iκβ kinase (PKA/PP2A/IKK) axis that in turn maintains initiator caspase8 activity. Lysosomal distribution of tBid stimulates cytosolic cathepsin B resulting accumulation of truncated-AIF with induction in scramblase mediated phosphatidylserine exposure in HepG2CR cells. Andrographolide treatment thereby switch on subG1 phase arrest by modulating cellular check points (cyclin A, B, cyclin dependent kinase-1) cueing to the apoptosis event. Collectively, this study suggested antineoplastic potential of andrographolide through PKA/PP2A/IKK pathway in HepG2CR cells.

Introduction

mesenchymal transition (EMT), hypoxia-inducible factor1-α (HIF1-α) signaling and DNA damage repair govern MDR induction, in chemo-resistance of HCC (Wen et al., 2016). Combined chemotherapy based on cisplatin, recommended by international cancer organizations has become a potential line of chemotherapy against liver cancer in recent times (Buendia and Neuveut, 2015) and continued to be a mainstay to treat HCC (Kim et al., 2017). Widespread use of platinum drugs led to a gradual design of escape route for tumor cell to build up resistance that reduces the effect of chemotherapy to a significant extent developing intense modifications at both molecular and cellular levels about cell survival/death, endocytosis, gene activation/silencing by regulating methylation and acetylation as well as mutations mediated by transcription factors/miRNAs (Shindo et al., 2018). Hence, the concept of using phytomedicines warrants immediate attention to overcome drug resistance.
Protein phosphatase 2A (PP2A) play dual role in keeping both pro-survival as well as proapoptotic signaling networks in check, maintaining a crosstalk with protein kinase A (via mitogen activated protein kinase signaling) and mediates Iκβ kinase (IKK) inhibition (Janssens and Rebollo, 2012) either under basal conditions (in the absence of a pro-survival signal) or serves in a negative feedback loop–constituted by the early nuclear factor –κβ (NF- κβ)-mediated re-synthesis of Iκβ (an NFκβ target gene) – that allows re-accumulation of Iκβ and termination of NF- κβ-activity (Barisic et al., 2008 ; Witt et al., 2009). It is to be noted in this context that PP2A-substrate-inhibitor interaction further potentiates several adenosine triphosphatedependent drug efflux pumps (MDR proteins) (Krauß et al., 2008).
Many studies have suggested chemotherapy with andrographolide as a single agent or in coadministration with cisplatin as a prospective therapeutic strategy in several types of cancer cells (Mishra et al., 2015; Lin et al., 2014; Zhou et al., 2012) which might be mediated via activating both intrinsic and extrinsic apoptotic pathways (Lin et al., 2014). Previous studies documented many bicyclic diterpenoid lactones like abitene, paclitaxel, docetaxel which functions through the modulation of PP2A/calcium unit (Yang and Dou, 2010; Wang et al., 2017). These findings cue to assess the efficacy of andrographolide through PKA/PP2A/IKK axis in the induction of cytotoxicity in cisplatin-resistant human hepatocarcinoma (HepG2CR) cells. Altered biology of scramblase narrated herein persuades the tale of intrinsic potency in lysosomal de-stabilization of HepG2CR cells. The results demonstrated that accumulation of cyclic adenosine monophosphate (cAMP) may potentiate extrinsic pathway of apoptosis by modulating cathepsinB-tAIF-scramblase activities after andrographolide treatment in cisplatin-resistant HepG2 cells.

2. Materials and Methods

2.1 Preparation of drug resistant cell line and reagents

HepG2 cell line was collected from Indian Institute of Chemical Biology (Kolkata, West Bengal, India), originally purchased from ATCC, Manassas, VA, USA. Cell line was treated in pulse, at IC50 of cisplatin (7.08 ± 0.073 μg/ ml) for 4 to 6 hour (hr) for developing cisplatin resistant HepG2 cells (HepG2CR) (Sakina et al.,2007). After six complete cycles of induction, selected cells were maintained in drug-free RPMI 1640 medium (Life Biosciences, Gibco, Walham, MA, USA) containing 10% fetal bovine serum (FBS) (Gibco-Thermofisher Scientific, Walham, MA, USA), 1% penicillin-streptomycin mixture (Sigma Aldrich, St. Louis, MO, USA) and 0.1% Fungizone (Sigma Aldrich, St. Louis, MO, USA) at 37°C under 5% CO2 containing humidified atmosphere (Sengupta et al., 2017) and grown to 70%-80% confluence. Cells completing this paradigm were termed HepG2CR. Further experiments were performed after maintenance of the cell line for 4 weeks in drug-free medium. Required chemicals unless otherwise mentioned were purchased from Sigma-Aldrich Corp., MO, USA.

2.2 Drug treatment

HepG2CR cell line was treated with 50μM cisplatin, andrographolide (40 μM)-cisplatin combination and a series concentration of (0.1 – 100) μM andrographolide for 12, 24, 48 and 72hr for further experiment. HepG2 cells were treated separately with 40 μM andrographolide for the similar time frame.

2.3 Cell survivability detection by water soluble tetrazolium (WST) assay and crystal violet staining

HepG2CR cells (1x104cells per well) were incubated in a 96 well microtiter plate for 18-24hr.The cells were then treated with 0.1,1,10, 20, 40, 80 and 100μM andrographolide as well as with 50μM cisplatin alone and in combination with 40μM andrographolide for 12, 24, 48 and 72hr respectively. Following the same protocol HepG2 cells were also incubated with 40μM andrographolide. 10μl WST-1 reagent was added followed by 0.5-4hr incubation at 37ºC in 5% CO2. Absorbance was measured at 420-480nm comparing reference value of 650nm using a 96 well microplate reader (BioRad Laboratory, Hercules, California, USA) according to the protocol of Roche Diagnostics Corporation, Indianapolis, USA (Sengupta et al., 2017). To revalidate cell survivability findings, crystal violet staining was done with same concentration of cells. Briefly, 50μl staining solution was added to each well and incubated for 20min at room temperature. After thorough washing solubilization solution was added and incubated again for 20min at room temperature with a frequency of 20 oscillations per minute. Optical density of each well was determined at 595nm (Kit No#ab232855, Abcam, Cambridge, UK). Data were represented in percentage of viable (attached) cells calculating against the values of untreated set (Feoktistova et al., 2016).

2.4 Immunoblot analysis

HCC cells were homogenized in lysis buffer and protein concentration was measured by the Folin-Ciocalteu Lowry method (Roura et al., 2006). Protein solutions (120 μg/ μL) were separated using 12% SDS-PAGE and transferred to nitrocellulose membrane (Millipore, MA, USA). Membranes were blocked in 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for 12h at room temperature and then incubated at 4°C with anti Cyclin D1, anti Cyclin E, anti Cyclin A, anti Cyclin B, anti p-Tyr15 Cdk1, anti p-Thr161 Cdk1, anti cFLIP, anti Iκβ, anti AIF/tAIF and anti tBID primary antibodies (SantaCruz Biotechnology, Dallas, Texas, USA), separately, at a dilution of 1:1000-1500 for 6hr at 4ºC according to manufacturer’s protocol. Membranes were then incubated with appropriate antimouse and anti-rabbit AP conjugated secondary antibody (1:2000; SantaCruz Biotechnology, CA, USA) in TBST for 2.5hr. Binding signals were visualized with NBT-BCIP substrate (Sengupta et al., 2017). Respective band densitometric analysis was carried out with ImageJ software (NIH, Bethesda, MD, USA). Protein loading was normalized using an anti-LAMP1, anti-VDAC1-porin and anti-β-actin, anti-KDEL (1:5000, Santacruz Biotechnology, CA, USA) for nuclear and cytoplasmic localization respectively.

2.5 Flow cytometric analysis

To investigate the cell cycle and programmed cell death in HepG2CR and HepG2 cells flowcytometric analysis was performed using propidium iodide (PI) and annexinV-FITC with PI, respectively. Cells were counted in different phases of cell cycle by analyzing PI fluorescence flowcytometrically at 488nm (Pozarowski and Darzynkiewicz, 2004) using fluorescence activated cell sorting (FACS; FACSCalibur (BD Biosciences, Mountain View, CA, USA).
Hepatocellular carcinoma cells were diluted to 1:100 (5 × 106cells/ml) by binding buffer, pH 7.4 containing 10mM HEPES–Na, 136mM NaCl, 2.7mM KCl, 2mM MgCl2, 1mM NaH2PO4,5mM glucose, 5mg/ml BSA and 2.5mM CaCl2. 1μg/ml AnnexinV-FITC and PI were then added to each of the samples follwing FITC- Annexin V appoptosis detection kit. (Cat. no. 556547, BD Pharmingen, CA, USA). Fluorescence intensity was then measured by FACS after 15min as described by Nicolay et al., 2008. Percentage of dead cells was calculated using CellQuest software (BD Biosciences, CA, USA) attached with the flow-cytometer.

2.6 DNA fragmentation analysis

Treated and untreated cells were washed with PBS, lysed with DNA lysis buffer and incubated (20mM EDTA, 100mM Tris, pH 8.0, 0.8% SDS) for 20min at room temperature. Samples were micro-centrifuged at 8000g for 15min and the supernatants were treated with RNase A (500μg/ml) for 30min at 37°C, followed by digestion with 500μg/ml proteinase K for 2hr at 55°C. DNA was extracted by using standard phenol chloroform method as described (Tee et al., 2012). Extracted DNA was resolved in 1.2% agarose gel and visualized with ethidium bromide for DNA fragmentation analysis (Lee et al., 2012).

2.7 Estimation of scramblase activity

Scramblase activity was assayed as previously described (Sahu et al., 2008). Briefly, 106 cells were loaded with 2.5-3μM 1-palmitoyl-1-[6-[(7-nitro-2–1, 3-benzoxadiazol-4-yl) amino]caproyl]sn-glycero-3 phosphocholine (C6 NBD-PC) were suspended in HEPES buffer, pH 7.4, containing phenylmethylsulfonyl fluoride (PMSF) and incubated at 37°C for internalization.
NBD-PC labeled lipid was removed from the outer layer of the membrane by back extraction and the pellets were solubilized in 1% (w/v) TritonX-100. The amount of internalized phospholipid was determined by comparing the fluorescence intensities before and after back exchange at an excitation wavelength of 470nm and emission wavelength of 540nm using HepG2CR cells were centrifuged at 15000g for 15 min with cathepsin B lysis buffer to collect cytosolic extract as enzyme source. Cathepsin B reaction buffer and amino-4-trifluoromethyl coumarin (Ac-RR-AFC) was added in equal proportion in order to achieve the fluorescence at excitation wavelength /emission wavelength (Ex/Em) of 400nm/ 505nm of released AFC (amino-4-trifluoromethyl-coumarin). The assay was carried out by using Cathepsin B activity assay kit (Fluorometric, Abcam, Cambridge, UK) according to Zou et al., 2017 and was represented in relative fluorescence unit (RFU).

2.10 Lysosomal integrity assay

Treated and untreated cells were exposed to acridine orange (AO; 5μg/ml) or lysotracker red (25nM) for 30min at 37°C. The cells were then washed with PBS and green cytosolic as well as red lysosomal fluorescence+ cells were determined by flow cytometric analysis. Lysotracker red retention or AO green and AO red fluroscence+ cells were determined by measuring 10,000 cells per sample using FL1 channel (FACSVerse; BD Biosciences).

2.11 Caspase8 activity analysis

Enzymatic activity of caspase 8 was measured by a FITC kit (Biovision Inc. Mountain View, CA, USA) as per Ohashi et al., 2013. Briefly Trypsin-EDTA treated 2.5×105 cells were incubated with 1μl FITC-IETD-FMK at 37°C for 0.5-1hr. Cells were then resuspended in 100μl wash buffer and fluorescence intensity was determined at Ex/Em: 485/535nm. Data were represented in FITC fluorescence in arbitrary units (AU).

2.12 Electrophoretic mobility shift assay (EMSA)

For performing EMSA, oligonucleotides for NF-κB:5’-AGCTTGAGGGGACTTTCCCAGGC3’ was end-labeled byT4 nucleotide kinase in presence of [γ32P] ATP. Nuclear extract was incubated with 0.05pmol radio-labeled oligonucleotide and poly (dl•dC) in a buffer containing 10mM Tris-HCl (pH 7.5), 1mM MgCl2,0.5mM EDTA, 0.5mM dithiothreitol, 50mM NaCl, 4% glycerol and 0.1% Nonidet P-40. Finally, samples were resolved in polyacrylamide gel electrophoresis (PAGE) and radioactive bands were quantified with Image J (NIH) to determine band shifting (Holden and Tacon, 2011)

2.13 Iκβ kinase (IKK) activity assay

IKK activity was assessed as per Kim et al., 2011 using human IKK and  kinase assay/inhibitor screening kit (Cat # KA0078 V.01, Abnova, Taiwan). Briefly, 10μl cell extract and 90μl kinase reaction buffer (kinase buffer: 20X ATP:19:1) were added to recombinant Iκβ substrate (KKKERLLDDRHDSGLDSMKDEEYE) pre-coated microwells. Phosphorylation of substrate was estimated by anti-phospho-IκβαS32 monoclonal antibody AS-2E8 as primary, antimouse-IgG-HRP+as secondary and tetra-methylbenzidine as chromogenic substrate. Absorbance values in ELISA were compared as AU at 450nm.

2.14 PP2A phosphatase activity assay

Cell lysates were incubated with PP2A capture antibody coated microtiter plates. Next, plates were treated with 200μM serine/threonine phosphatase substrate (DLDVPIPGRFDRRVS(PO3)VAAE) in presence of malachite green reagent A and B. Green coloured product of the reaction was estimated by ELISA plate reader (Model 680, Biorad Laboratory) at 620nm. Values were represented in nmole phosphate released/min/mg protein for comparison. Wells devoid of either lysate or enzyme served as background measurements.

2.15 Protein kinase A (PKA) activity assay

Enzymatic activity of protein kinase A was estimated as per Ruiz-Ojeda et al., 2016 by Enzo life science kit (ADI-EKS-390A). Cell lysate was added to PKA substrate coated microtiter plate and reactions were initiated by addition of ATP, wells were incubated with phosphospecific substrate antibody as primary and HRP conjugated anti-rabbit-IgG as secondary antibody after thorough wash. 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was added to measure absorbance in ELISA reader at 450nm.

2.16 Quantitative estimation of cAMP

cAMP content was measured following fluorometric cAMP direct immunoassay kit (Enzo life science, ADI-901-066) according to Xu et al., 2013. Cell extracts were added to anti-cAMP antibody coated plate and were incubated with 1x HRP-cAMP working solution. Then extract was washed and 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) as well as H2O2 were added. Generated resorufin fluorescence (Ex:540nm/Em:590nm) was estimated and values were represented in pmol/mg protein against reference graph of standard cAMP.

2.17 Statistical analysis

All data was evaluated in duplicate against HepG2CR cells and collected from at least 5 independent experiments, unless otherwise indicated. Data were graphed and analyzed using GraphPad InStat software (Graph Pad, La Jolla, CA, USA) using one-way ANOVA followed by Dunnet’s post hoc analysis and student’s unpaired t-test where appropriate. p< 0.05 is considered as statistically significant among treatment group/s. 3. RESULTS 3.1 Andrographolide treatment document reduced cell survivability To understand the effect of andrographolide on HepG2CR cell viability and establish the degree of cytotoxicity due to the phyto-extract, HepG2CR cell line was incubated for 12, 24, 48 and 72 hours with 50μM cisplatin (IC50) and/or a range of concentration (0.1 -100) μM andrographolide. Following analysis of cell viability by WST-1 assay, 100% survivability was observed after cisplatin treatment (Figure 1A, B, C, D). Andrographolide regimen (40μM) cue at a significant initiation of decreasing trend in cell survivability (~80%) after 12hr (Figure 1A) and most specifically during 24hr (~70%), (Figure 1B), 48 hr (~50%) (Figure 1C) of subsequent incubation. Extension of the treatment for further 24hr indicated a marked decrease in cell survivability after andrographolide regime which was followed thereafter by more distinct reduction in cell survivability after 80 and 100μM treatments (Figure 1D). Distribution of cell survivability in the treated groups after 12, 24, 48 and 72hr of incubation suggested commencement of the reduction of ability to survive cells to 50% specifically after 48hr of treatment with 40μM andrographolide (Figure 1E). However, a combination treatment of cisplatin-andrographolide on HepG2CR cells at different time points (12, 24, 48, 72 hr) showed no significant difference (Figure 1A, B, C, D) against respective individual andrographolide treatment. While andrographolide alone exposure on HepG2 demonstrated a reduction in cell survability at 24 (Figure 1B), 48 (Figure 1C) and specifically after 72hr (Figure 1D) against andrographolide treated HepG2CR. Crystal violet staining suggested significant reduction in the attached cell population in a range ~50% after andrographolide alone (40μM) and in combination with cisplatin (50 μM) treatment for 48hr. ~40% cell population remained in attached form after 48hr of 40μM andrographolide treatment to HepG2 cells (Figure 1F). 3.2 Effect of andrographolide on cell cycle regulation Previous studies reported by application of siRNA profiling, transcription of cell regulatory proteins was dysregulated in cisplatin-resistant cells (Shen et al., 2012). To further verify this result, we analyzed the protein levels of cell cycle by immnoblot analysis. Cyclin D1 and cyclin E expressions were comparable after cisplatin and/or andrographolide treatment with respect to untreated HepG2CR (Figure 2A, B) cells. However, 40μM andrographolide treatment for 48hr documented a significant decrease (p<0.01) of cyclin A (Figure 2C) and cyclin B (Figure 2D) in HepG2CR cells, which further down-regulated with an increase in the drug concentration. Cdk1 activity, a central regulator of cell cycle that drives cells through G2/M phase, is dependent upon phosphorylation at tyrosine15 residue leading to inhibition of the enzyme while it gets activated by the phosphorylation at Thr-161 residue (Wang et al., 2015). Significant increase (p<0.01) was observed in p-Tyr15 (Figure 2E) with a concomitant decrease (p<0.01) of p-Thr161 after exposure of 40μM andrographolide on HepG2CR cells (Figure 2F). Extent of phosphorylation at inhibitory site was further enhanced (Figure 2E) in parallel to reduction in stimulatory phosphorylation (Figure 2F) Hence, andrographolide treatment may prompt a possible amelioration of cell cycle in HepG2CR. 3.3 Andrographolide treatment causes subG1 phase arrest in resistant cells HepG2CR cells were treated with 50m cisplatin and/or series concentration (0.1 -100) m of andrographolide for 48hr to carry out cell cycle analysis. The results showed that proportion of apoptotic cells in subG1 phase was significantly less in groups treated with/without cisplatin when compared to andrographolide alone (40μM) (p<0.05) (Figure 3A). SubG1 phase arrest was documented after andrographolide (35%) and co-treatment of cisplatin-andrographolide (39%) in HepG2CR cells suggesting no appreciable change in either exposure. 40M andrographolide treatment documented increased cell number thereafter with respect to concentration (Figure 3B). As shown in Figure 3C, a gradual decrease of cell count after andrographolide regimen was observed suggesting reduction of cellular turn over in G1 phase. Increased cell number at S phase suggested a mere enhancement in DNA replication with the series increase in drug concentration, which may be a possible protection tried over drug exposure (Figure 3D). An initial significant enhancement (p< 0.05) in the cell population after 0.1μM andrographolide exposure experience a sudden thwart at 10M and document a declining plateau. Combined treatement of andrographolide with cisplatin nonsignificant change in G1 (Figure 3C), S (Figure 3D) and G2 (Figure 3E) phase against respective andrographolide treatment. Andrographolide treatment to HepG2 cells exhibited effective increase in subG1 (Figure 3B), decrease in G1 (Figure 3C), increase in S (Figure 3D) and decrease in G2 (Figure 3E). These results demonstrate that andrographolide increase the sensitivity by retaining cells in subG1 phase. 3.4 Andrographolide treatment initiated apoptosis through increased scramblase activity Increase in subG1 peak after andrographolide treatment is an indication of the loss of DNA during a probable programmed cell death. An isolated cell population as high as 50.12% annexinV-FITC+ was observed after 40μM andrographolide treatment (Figure 4A), however, which was increased to some extent with the increase in dose to 80 and 100μM andrographolide (Figure 4B). This was further supported by the documentation of smear associated with probable double strand breaks in andrographolide alone and cisplatin treated HepG2CR cells and andrographolide treatment in HepG2. DNA fragmentation remained arrested even after cisplain treatment suggesting effetive establishment of drug resistance in HepG2CR (Figure 4C). Cisplatin treatment increases the percentage of cells to a smaller degree (3.89%) but the significant increase (p< 0.05) commenced from 0.1μM andrographolide regimen with respect to HepG2CR cells (1.1%). However, there was no significant difference between individual andrographolide (40μM) (~51%) with a co-treatment of andrographolide and cisplatin on HepG2CR cells (~56%) while a certain decrease was observed in andrographolide treated HepG2 (~61%) cells (Figure. 4B). Phospholipid scramblase function is enhanced during both cell stimulation and apoptosis (Frasch et al., 2000). Activation of the scramblase leads to exposure of phosphatidylserine as well as simultaneous internalization of phosphatidylcholine with respect to membrane surface. Therefore, amplified annexinV-FITC fluorescence instigated us to carry out the experimental analysis on scramblase activity by incubating the cells with NBD-PC followed by determination of internalized NBD fluorescence as a marker of enzymatic activity. Data analysis displayed more or less similar percentage of NBD-PC internalization during cisplatin (50μM) treatment with respect to untreated group even after 60min of NBD-PC incubation. 0.1μM andrographolide mostly after 40 min, 1μM andrographolide after 20 min and from 10μM onwards the values were significantly higher in each and every point of time frame with respect to HepG2CR. Treatment with 40μM andrographolide demonstrated a sharp increase in percentage of NBD-PC internalization and further increase in doses to 80 and 100μM suggested a leveling up of the process in the drug resistant group (Figure 4D). 3.5 Andrographolide regime down-regulates lysosomal integrity through cathepsin B –tAIF pathway in HepG2CR cells Activity of the scramblase is dependent upon intracellular calcium concentration and cytoplasmic localization of tAIF. Ratiometric fluorescence value at 340:360 nm demonstrated no significant change after cisplatin (50µM) and a series concentration of andrographolide (0.1100µM for 48hr) regimen (Figure 5A). Data suggested more or less equivalent intracellular calcium concentration documenting andrographolide treatment which probably did not play any role in modification of scramblase activity. A sequential increase in the intensity of cytosolic tAIF was however observed from 0.1 to 20µM andrographolide treatment which was significantly increased (p<0.01) after 40µM and thereafter (Figure 5B). Mechanisms that cause the release of tAIF during andrographolide treatment have not been yet determined. During death inducing insult, calpains or cathepsins cleave AIF to produce tAIF which is released from mitochondria to cytosol for its categorical function (Rodriguez and Torriglia, 2013). Activity of calpain is dependent upon calcium concentration that remained unchanged after drug treatments (Figure 5A). Data analysis indicated a serial significant increase (p<0.05) in cytosolic cathepsinB activity from 0.1 to 20μM andrographolide treatment with a sharp spike in the enzymatic activity after 40 μM (p<0.01) and subsequently (Figure 5C). Increase in cytosolic cathepsin B activity indicated its release into cytoplasm from lysosome. Flow-cytometric analysis displayed a significant reduction (p<0.01) in AO-specific metachromatic red fluorescence+ cells after andrographolide treatment (0.1 to 100μM) sequentially (Figure 5D). 40μM andrographolide depicted leftward shifting with only 32.9% positive cells after 48hr of treatment (Figure 5E). Increase in concentration upto 80 and 100μM andrographolide further reduced AO red positive cells as suggested by the experimental analysis (Figure 5D). Non-cumulative AO staining illustrated increase in green fluorescence positive cells along the course of andrographolide regime (0.1 to 100μM) whereas lysotracker red accumulation within lysosomes reduced gradually (Figure 5D). Treatment with a concentration of 40μM andrographolide demonstrated rightward shifting of histogram with 77.13% AO green positive cells after 48hr (Figure 5E). According to the data 21.88% cells were lysotracker red positive after 40μM andrographolide treatment for 48hr (Figure 5E). Cisplatin treatment did not show any significant effect since they demonstrated 88.7% AO red positive cells with respect to 94.59% in HepG2CR, 17.01% AO green positive cells with respect to 3.67% in HepG2CR and 75.24% lysotracker red positive cells with respect to 92.6% HepG2CR indicating drug resistance and maintenance of lysosomal stability as observed by the study (Figure 5E). 3.6 Andrographolide treatment augment translocalization of tBid to instigate caspase 8lysosome mediated apoptosis Reduction in the retention of lysotracker and polymeric AO aggregate suggested lysosomal membrane permeabilization. tBid homo-oligomers are reported to associate with lysosomal membrane permeabilization (Zhao et al., 2012). In our experiment lysosomal fraction was isolated and was blotted against anti-VDAC1-porin (as marker of mitochondria), anti- KDEL sequence (as marker of endoplasmic reticulum) and anti-LAMP1 (as a marker of lysosome) antibodies to determine the purity of the fraction. Presence of protein bands against anti-LAMP1 suggested successful separation of lysosomal fraction (Figure 6A). Immunoblot analysis indicated significant enhancement (p<0.05) in tBid expression in lysosomal fraction after 1, 10 and 20μM andrographolide treatment sequentially (Figure 6B). Data represented a significant (p<0.01) translocalization of tBid to lysosome after 48hr of 40μM andrographolide treatment (Figure 6A) which maintained a plateau after 80 and 100μM regimen (Figure 6B). Considering the role of caspase8 in truncation of Bid in parallel to increased content and allocation of tBid to the lysosome, caspase8 activity was analyzed. Study suggested increased activity of caspase8 with simultaneous increase in doses of 0.1 to 100μM andrographolide suggesting caspase 8 mediated lysosomal destabilization. However, a marked amplification in the activity was observed at 40μM andrographolide and thereafter (Figure 6C). Cellular caspase-8 (FLICE)-like inhibitory protein or cFLIP, the inhibitor of death-receptor signaling, forms heterodimer with caspase8 and helps to sustain cell survival and proliferation (Kavuri et al., 2011). Data suggested a reduction in cFLIP expression with increasing doses from 0.1 to 20μM andrographolide along with a significant reduction (p<0.01) after 40μM andrographolide (Figure 6D). Cisplatin treatment did not show any significant alteration in lysosomal localization of tBid (Figure 6B), caspase8 activity (Figure 6C) and cFLIP expression (Figure 6D) with respect to cisplatin resistant HepG2 cells. 3.7 Andrographolide treatment decreases nuclear NFκβ prompting the beginning of the end Constitutive activation of the NFκβ pathway is a feature of hepatoma (Li et al., 2010). Being a transcription factor NFκβ also induces cFLIP expression after its translocation to the nucleus following activation (Ranjan and Pathak, 2016). EMSA analysis with NFκβ specific probe suggested a distinct band shifting both in HepG2CR and cisplatin treated (50μM) HepG2CR cells indicating presence of NFκβp65 subunit within the nucleus. A progressive reduction (p<0.05) was observed in the shifted band intensity after andrographolide treatment (1 to 20μM) which have reached the peak at 40μM (p<0.01) onwards (Figure 7A). Restraining of NFκβ from its nuclear translocalization is dependent upon persistent concentration of inhibitor of κβ (Iκβ) within cytoplasm. Immunoblot analysis revealed an increase in the protein expression with the increase in concentration of andrographolide (1 to 100µM) (Figure 7B). The quantal dose of 40µM again stood apart depicting the start of growth plateau documenting significant amplification (p<0.01). Data specified that andrographolide treatment reduced NFκβ nuclear translocation by retaining them within cytoplasm after their probable binding with increased concentration of Iκβ during treatment. IKK complex is the signal integration hub for NF-κB activation. Phosphorylation followed by proteasomal degradation of Iκβ helps in the nuclear localization of NF-κβ and potentiates cell survivability (Zheng et al., 2017). The increase in band intensity of Iκβ prompted us to investigate the activity of IKK after andrographolide treatment. ELISA analysis displayed reduction in enzymatic activity with the increase in doses from 0.1 to 20μM andrographolide. However, a definite suppression of the activity was observed after 40μM andrographolide treatment for 48hr which down regulate gradually (Figure 7C). Recent studies demonstrated activation of PP2A by a cAMP/PKA-dependent pathway (Ahn et al., 2007) and a potential of developing apoptosis in cancer cells by dephosphorylating IKK. ELISA analysis resulted a gradual increase in PP2A (Figure 7D) and PKA (Figure 7E) activity after andrographolide exposure (1 to 100 µM). Effective concentration of 40µM andrographolide indicated a significant increase (p<0.01) in both the cases demonstrating a possible reason for decrease in cell survivability after andrographolide regimen. Increased activity of protein kinase A after andrographolide treatment suggested for the determination of intracellular cAMP content before and after drug treatment. Estimation of resorufin fluorescence followed by its comparison against standard curve portrayed increasing trend of cAMP accumulation in parallel to rise in doses from 0.1 to 20μM andrographolide during treatment Effective concentration of 40μM andrographolide exposure significantly increased (p<0.01) cAMP content in HepG2CR cells. Enhancement of doses upto 80 and 100μM andrographolide depicted boosting of only a mere content of cAMP (Figure 7F). 4. Discussion: Drug resistance is one of the main hurdles to overcome effective therapy along with individual side-effects, which warrant an approach of identification of bio-molecule/s for a direct action, or to bypass or overcome resistance to established drugs for effective treatment. In this report, we demonstrate that cathepsin B mediated scramblase activation incur altered lipid asymmetry at the level of cell membrane in HepG2CR cells by modulating PKA/PP2A/IKK axis after andrographolide exposure causing subG1arrest. The scramblase activity gained its momentum from the accumulation of tAIF in the cytoplasm and as apoptosis is an irreversible event, sustained activation of scramblase allowed for maximal phosphatidyl serine (PS) externalization (Figure 8). In this context, we have also revalidated the hypothesis that enhanced scramblase activity is required for surface PS exposure (Figure 4). The underlying rationale for the usage of andrographolide is the understanding of the heterogeneous drug-resistant tumor clones, especially in the context of HCC. Andrographolide targets TLR4/NF-κB/MMP-9 pathway leading to inhibition of proliferation of human colon cancer (Zheng et al., 2017) and lung cancer through the suppression of TGFβ1/VEGF/protein kinase C (Luo et al., 2014) According to the citations, 4 μg/mL or 10 μM is considered as the upper IC50 limit for a promising cytotoxic compound after incubation for 48 and 72hr (Kuete and Efferth 2015; Kadioglu et al., 2018). In the present work, HepG2CR cells although crossed the threshold value, were treated in pulse for only 4-6hr duration. WST-1 assay showed that proliferation of HepG2CR cells at 40M concentration was suppressed (Figure 1) which was revalidated by crystal violet staining. These data show the anti-proliferative potential of andrographolide against drug-sensitive and -resistant cancer cell lines since identification of compounds able to overcome MDR is an attractive strategy in drug research (Gillet et al., 2007). Cell survivability is intrinsically associated with cell cycling. Recent studies have highlighted cell cycle arrest especially in G2/M phase in melanoma cells (Liu and Chu, 2018) and breast cancer cell line (Banerjee et al., 2016). Our study showed a decreased cell proportion in sub G1 phase in HepG2CR cells indicating requirement of DNA replication to protect cells under stress during treatment. However, G2/M phase displayed increase in cell count at low dose (1μM) and values decreased thereafter in a sequential manner. In need to understand the possible underlying mechanism, we documented an imposing effect of andrographolide by modulating specific cyclin and cdk activities. Data reflected similar quotient of cyclin D1 and cyclin E in quantity after any of the doses of drug treatment. Cyclin A and/or B, the subG1 and G2/M phase cyclins were significantly reduced in HepG2CR cells after andrographolide treatment which explains perturbed mitosis. Further analysis also suggested enhanced inhibitory phosphorylation at Tyr15 residue and simultaneous decreased phosphorylation at Thr161 residue resulting permanent inhibition of mitosis promoting factor. Activation of Cdk1 requires synthesis and accumulation of cyclin B, binding of cyclin B to Cdk1, and removal of the inhibitory tyr-15-Cdk1 phosphorylation (Huang et al., 2013). Similar findings have also been reported by others using myeloma cells, HepG2 cells, (Hedblom et al., 2013, Chaudhary et al., 2013) etc. FACS analysis with annexinV-FITC corroborated the idea of programmed cell death since a high percentage of cells were FITC+ in 1, 10, 20μM and specifically after 40μM (50.12%) of treatment (Figure 4). After 48 hr treatment in HepG2CR with 40 M andrographolide and a co-treatment of cisplatin and the referred bio-molecule, inter-nucleosomal DNA fragmentation was found which is considered as one of the biochemical hall mark of apoptosis, as shown in Figure 4. Enhanced scramblase activity is an important feature during apoptosis which have simultaneous events of chromatin condensation and DNA fragmentation (Frasch et al., 2000). A significant enhancement of the enzymatic activity specifically after 40μM and onwards andrographolide treatment indicate a possible exposure of PS and internalization of phosphatidylcholine by jumbling of plasma membrane. Observation itself was an explanation for increased binding of annexinV which predominantly interacted with exposed PS and gave enhanced fluorescence of FITC in the andrographolide treatment reflecting higher rate of cell death. Considering activation of scramblase as a major event detailed mechanistic analysis was conducted by estimating intracellular calcium concentration since calcium is an effective regulator of this enzyme activity. No significant change in calcium concentration in treated group directed the study towards determination of the status of cytosolic truncated apoptosis inducing factor (tAIF), the well known inducer of scramblase. Increased accumulation of tAIF in cytoplasm suggested a possible reason for the induction of scramblase in HepG2CR after andrographolide treatment. AIF is generally localized within mitochondria and only post truncation the translocation from mitochondria to cytosol is possible (Bano and Prehn, 2018). Truncation or cleavage of AIF is dependent upon calpain or cathepsinB protease activity (Rodriguez and Torriglia, 2013). Apoptosis, an efficient cell death program, is primarily mediated through the intrinsic or the extrinsic pathway in response to different stimuli in various cell types of which the latter is dominated by cFLIP, tAIF, cathepsin B, caspase 8, tBid to name a few. The downstream pathway of this signaling bifurcates to calpain and cathepsin depending on calcium potential (unchanged/ altered). Absence of change in calcium concentration and increased cathepsin B activity in our study ensue cathepsin B signaling after andrographolide treatment. CathepsinB is mostly localized within lysosome and it is released barely after lysosomal destabilization (Stoka et al., 2016). Flowcytometric analysis suggested accumulation of AO green monomers and loss of lysotracker red in HepG2CR after andrographolide treatment. Therefore, lysosomal instability probably played an instrumental role in tAIF signaling. According to data instability in lysosomes is dependent upon lysosomal translocalization of tBid. Bid protein was actually truncated by caspase8 which became activated only after andrographolide treatment following the previous report where death inducing signaling complex (DISC) activation and caspase8 induction were mentioned after chemotherapy with andrographolide (Dickens et al., 2012). Activity of caspase8 was retained due to reduction in cFLIP expression, the specific biological inhibitor of caspase8, as demonstrated by the band intensity analysis of the respective immunoblot in andrographolide treated group. cFLIP is targeted for cancer therapy considering its critical anti-apoptotic regulation through inhibiting TNF-α, FasL and TNF-related apoptosis inducing ligand (TRAIL) induced apoptosis (Kong et al., 2017). Expression of cFLIP is significantly up-regulated by NFκβ when it is permitted to translocate to the nucleus (Ranjan and Pathak, 2016). Thus absence of NFκβ in nuclear fraction was the technical explanation for reduced cytosolic cFLIP content after drug treatment. Investigation prompted us to analyze the status of Iκβ after andrographolide treatment which suggested an increased accumulation of the protein post therapy with respect to HepG2CR, in presence or absence of cisplatin (Figure 7). Iκβ stability is dependent upon its IKK mediated phosphorylation, a prerequisite for the proteasomal degradation (Gringhuis et al., 2005). Result indicated reduction in IKK activity providing a suitable explanation against Iκβ accumulation following andrographolide treatment. Activation of IKK is suppressed by its protein phosphatase2A (PP2A) mediated dephosphorylation (Tsuchiya et al., 2017). In our study, a definite enhancement in the PP2A activity with an indication of its significant role in the reduction of IKK activity after drug treatment was observed. Activation of PP2A is totally dependent upon protein kinase A (PKA) mediated phosphorylation (Ahn et al., 2007) which is supported by the increased PKA activity in the treated group. PKA activity is upregulated and sustained by the cAMP, the potential secondary messenger in cell biology. Andrographolide treatment effectively enhanced cAMP accumulation and that possibly in turn induced programmed cell death following the above said pathway in the cisplatin resistant HepG2 cell during experimental analysis. Therefore, andrographolide associated caspase8 activity-dependent cathepsinB-tAIF-scramblase action can be considered as an effective strategy for the introduction of programmed cell death even in the drug resistant cancer. The process is probably further potentiated by inhibition of PTEN, the effective endogenous inhibitor of Akt-mTOR pathway (Mi et al., 2016). As a result, cisplatin mediated induction of autophagy, the reported reason for drug resistance, can be arrested in parallel to generation of ‘eat me’ signal after the therapy with andrographolide. Digging into the knowledge of herbal medicine may uncover new leads for anti-cancer drugs. 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