Targeting Phosphatidyl-inositol-4-Phosphate-5-Kinase ameliorates hepatic cancer by inhibiting PI3K/Akt/mTOR and autophagy mechanism and enhancing ROS-mediated apoptosis

doi:10.21203/rs.3.rs-3967312/v1

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Research Article

Targeting Phosphatidyl-inositol-4-Phosphate-5-Kinase ameliorates hepatic cancer by inhibiting PI3K/Akt/mTOR and autophagy mechanism and enhancing ROS-mediated apoptosis

https://doi.org/10.21203/rs.3.rs-3967312/v1

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Background

Hepatic cancer cells control Reactive Oxygen Species (ROS) and lipid kinases to grow. PIP5K, a lipid kinase, modulates the proliferation and Autophagy; however, its role remains HCC progression is uncertain. This study examined the involvement of PIP5K in ROS-dependent autophagy-Nrf2 antioxidant pathways using α- and β-specific isoform inhibitors (ISA201IB and IITZ01) and discovered NG-TZ-17 and 20 as inhibitors under lead optimization from IITZ01.

Methods

PIP5K and its relationship with the ROS-Autophagy-Nrf2 axis were examined using western blotting and IHC in Hepatocellular carcinoma (HCC) tissue samples (n = 36) and hepatic cancer cell panels. To determine the role of PIP5K in ROS-mediated apoptosis, HepG2 cells (PIP5K highly expressed cancer cells) were treated with different amounts of H₂O₂ with and without PIP5K inhibitors and compared to a standard autophagy inhibitor. To support in vitro cell-based data, PIP5K inhibitors (IITZ01, 60 mg/kg and NG-TZ-17, 50 mg/kg) were orally administered for 10 days in a GFP-HepG2-induced hepatic cancer model in SCID mice. Animal imaging, tumor regression, survival, and protein expression in the isolated tumors were monitored.

Results

PIP5K isoforms, Beclin-1, and Nrf2 increased with HCC grade. Autophagy boosted the expression of PIP5K isoforms, Nrf2, HO-1, and SOD2, preventing peroxide-induced apoptosis. Under these conditions, PIP5K inhibitors increase ROS-mediated apoptosis by downregulating proliferation, autophagy, and Nrf2, indicating that PIP5K controls cellular proliferation, autophagy, and ROS-mediated apoptosis. In vivo research showed that PIP5K inhibitors (NG-TZ-17 and IITZ01) dramatically reduced the tumor burden in HepG2-xenograft SCID mice, comparable to sorafenib.

Conclusion

PIP5K isoforms induced hepatic cancer cell proliferation in response to ROS. Inhibition of PIP5K isoforms sensitizes hepatic cancer cells to ROS-mediated apoptosis by decreasing the PI3K/Akt/mTOR axis, autophagy, and Nrf2.

PIP5K

Hepatocellular carcinoma

PI3K/Akt

Autophagy

Reactive-Oxygen-Species

Hepatocellular carcinoma (HCC) contributes to approximately 700,000 cancer-related deaths annually, and a million liver cancer cases will be detected by 2025 (1). Alcohol-Associated Liver Disease (AALD), viral infections including Hepatitis B or C, Aflatoxin B1, genetic mutations, and Non-Alcoholic Fatty Liver Disease (NAFLD) are the major cause of HCC (2–4). Impairment of autophagy is a common mechanism in all risk factor-induced HCCs cases. Autophagy aids in cancer growth by promoting tumorigenesis and cell survival. Proliferative signals from various carcinogens overwhelm the evolutionarily conserved autophagy process for the quality control of cellular organelles and protein complexes, impairing autophagy and causing the accumulation of faulty oncogenic proteins that cause carcinogenesis. Once a tumor is established, elevated autophagy promotes metabolic and adaptive rewiring to sustain tumor proliferation and manage the tumor microenvironment by restoring nutrition, oxygen, and energy supply (5).

Multiple studies have shown a positive association between autophagy and proliferative markers as liver cancer advances, demonstrating that the PI3K and autophagy pathways act together and reinforce each other (1–3). High autophagy facilitates Nrf2 nuclear translocation, degrades Keap-1, and upregulates antioxidant enzymes. This allows cancer cells to grow under adverse conditions and promote resistance. Prolonged autophagy causes resistance to conventional and advanced chemotherapies such as VEGF, mTOR, and PDL1 inhibitors (6). Even the latest in-line PI3K/Akt inhibitors promote autophagy, which can lead to drug resistance and treatment failure. Hence, identifying a common target that plays a role in the proliferative and autophagy mechanisms may have dual benefits in treating cancer. PIP5K (Phosphatidyl-inositol-4-Phosphate-5-Kinase) is a target upstream of both proliferative (PI3K-Akt) and adaptive (autophagic) processes, especially late autophagic events; thus, blocking it inhibits both processes (4, 5). In the current study, we aimed to explore the role of PIP5kinase in the progression of hepatic cancer and its association with cell survival using PIP5K isoform-specific inhibitors using in vitro and in vivo models.

Tissue microarray slides were obtained from the DBT-National Liver Disease Biobank (NLDB) facility at the Institute of Liver and Biliary Sciences (ILBS) in New Delhi. The cell lines PLC/PRF5 (cat. CRL-8024), and SNU-387 (cat no. CRL-2237), and Skhep-1 (cat. HTB-52), and HepG2 (cat no. HB-8065) were purchased from ATCC. The primary antibodies used for immunohistochemistry, immunofluorescence, and western blotting were anti-mTOR (CST cat no. 2972), Phosphatidyl-inositol-4-Phosphate-5-Kinase 1A (PIP5K1A) (Abclonal cat no. A7749), and PIP5K1B (Abcam (cat. ab236114), p-Akt (CST, cat no. 9271), SRC (Abclonal, cat no. A19119), Beclin-1 (Abclonal cat. A21191), Nrf-2 (Abclonal cat no. A1244), SOD2 (Abclonal cat no. A1340), HO-1 (Abclonal cat no. A1346) and beta-actin (Abclonal cat. AC006). ISA-2011B was purchased from Life Technologies and Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit was purchased from Thermo Fisher (cat no.P36235) to establish stage-specific inhibition of autophagy. Normal goat serum was purchased from Cell signalling Technologies (CST, cat no. 5425) and prolong-gold antifade-DAPI (cat no. P36931), and prolonged gold anti-fade (cat. P36930) were purchased from Thermo Fisher Scientific. The GFP-cell labelling kit, Sorafenib, Chloroquine, ML-385, extracellular matrix, RIPA buffer, protease inhibitor cocktail, and phosphatase inhibitor cocktail were purchased from Sigma-Aldrich.

Expression levels of PIP5K and markers of autophagy in Hepatic cancer

The ILBS, New Delhi Biobank, provided tissue microarray slides of normal neighboring control and liver cancer specimens of grades 1–3. Sections were deparaffinized and rehydrated by serial treatment with xylene and ethanol at decreasing concentrations (100%, 95%, 72%, 50%, and 0% [cold tap water] ) for 3 min each. The sections were microwaved for 20 min in a citrate buffer (pH 6. After washing with Tris-buffered saline (TBS) 2–3 times, the slides were blocked with 5% bovine serum albumin (BSA) at room temperature for 2 h. The slides were treated with primary antibodies (1:200) against PIP5K1A, PIP5K1B, Beclin-1 (IF), Nrf2, and AKR1B10 at 4°C. The slides were rinsed with TBS after incubation with the primary antibody and treated with 0.3% H2O2 in TBS for 15 min for IHC. The slides were then treated with anti-rabbit HRP-conjugated secondary antibodies (IHC) and Alexa flor-488 conjugated antibodies (IF) (1:2000) at room temperature for 1.5 hours. The slides were then washed with TBS. IF slides were counterstained with Hoechst and placed on coverslips, whereas IHC slides were treated with DAB (chromogen) for 10 min and rinsed with tap water. For nucleus visualization, IHC slides were counterstained with hematoxylin. With 20X and 40X objective magnifications, the EVOS Auto-2 microscope captured IHC and IF bright-field and dark-field images. Picture J's IHC profiler scored IHC and assessed the fluorescence intensity of IF pictures. Hepatic carcinoma grade scores and fluorescence intensities were compared to those of the neighboring control tissue sections.

Cell culture

PLC/PRF5, SNU-387, Skhep-1, and HepG2 cells were cultured in Minimum Essential Media (MEM) (Gibco, Life Technologies) supplemented with 10% FBS and 1% antibiotic solution represented as complete media at 37°C in a humidified CO₂ incubator. The basal expression of PIP5K isoforms, autophagy, and Nrf2 in various hepatic cancer cells was explored in cells grown in complete medium.

Exploring the role of PIP5K in Reactive Oxygen Species (ROS) mediated apoptosis and autophagy

ROS act as upstream markers of cell proliferation, autophagy, and apoptosis. To study the impact of ROS on PIP5K, autophagy, and Nrf2, HepG2 cells were treated with increasing concentrations of H₂O₂ (6.25, 12.5, 25, 50, 100, and 200 µM) and cell viability was monitored using MTT at 48 h, both with and without serum. MitoSOX and lysotracker dyes were used to assess mitochondrial superoxide and lysosome levels in HepG2 cells treated with increasing H₂O₂ concentrations in serum-starvation medium. To understand how ROS levels affect proliferation, autophagy, and antioxidant gene expression, western blot analysis was performed for PIP5K1A, PIP5K1B, Beclin-1, Nrf2, Bax, and BCl-2. This was compared to cells grown in complete and starved media.

Development of PIP5K inhibitors

Previously, we synthesized and evaluated the cytotoxicity of benzimidazole-containing s-triazine molecules and identified IITZ01 (N2-[4-(1H-Bbenzimidazol-2-yl)phenyl]-N4-(4-fluorophenyl)-6-(4-morpholinyl)-1,3,5-triazine-2,4-diamine), which exhibited excellent cytotoxicity with moderate PI3K inhibition (7). Mechanistic evaluation has shown that autophagy is inhibited by the cytotoxic nature of the molecule (8). Upon further investigation of the molecular target underlying autophagy inhibition using a cell-free kinase inhibition assay, we observed that IITZ01 showed characteristic inhibition of the PIP5K1B isoform. IITZ01 at 1µM concentration exhibited 8%, 66%, and 35% inhibition of PIP5K1A, PIP5K1B, and PIP5K1C isoforms, respectively (data unpublished). To optimize the lead molecule IITZ01, we synthesized and screened biologically active IITZ01 derivatives for PIP5K inhibition, effect on cell viability of HCC cell lines (such as PLC/PRF5, SNU-387, Skhep-1, and HepG2), and autophagy inhibition (using acridine orange, GFP-RFP-LC3B-Premo autophagy sensor, and dual PIP5K-lysotracker dual staining assays).

Enzyme inhibition

Enzyme inhibition studies were outsourced to ThermoFisher Scientific, SelectScreen™ biochemical kinase profiling service using the Z-LYTE™ screening protocol and assay conditions. Biologically active derivatives IITZ01, IITZ02, NG-TZ-17, and NG-TZ-20 (1 µM) were incubated with a reaction mix containing phosphatidyl-serine (substrate), ATP, PIP5K1B enzyme, ADP, and ATP tracer antibodies for single point enzyme inhibition and later with serially three-fold decreasing concentrations starting from 1 µM till ten concentration points for 1 h, followed by the addition of labeled anti-ADP-AlexaFlor™ 647 antibody and EDTA to stop the reaction. Absorbance was measured and the IC₅₀s of active triazine derivatives were calculated from the concentration vs. percent inhibition graph.

Cell viability studies of PIP5K inhibitors on HCC cell lines

To estimate the IC₅₀ of IITZ01 and its derivatives, NG-TZ-17 and NG-TZ-20, in hepatic cancer cells, PRF5, SNU-387, Skhep-1, and HepG2 were plated into 96-well plates. Once the cells reached the desired morphology, they were starved for 2 h and then treated with serially increasing concentrations (0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, and 20 µM) of NG-TZ-17, IITZ01, ISA-2011B, and (1.25, 3.125, 6.25, 12.5, 25, 50, and 100 µM) of chloroquine (CQ). Thereafter, the cells were incubated for 48 h, followed by the addition of 10 µl of MTT (5 mg/ml), which was added to each well and incubated for 4 h. Thereafter, the MTT-containing solution was replaced with DMSO to dissolve the formazan crystals and the absorbance was measured at 577 nm. The percentage cell viability was calculated by dividing the absorbance of treated cells by the absorbance of untreated cells.

Establishing the phase-specific inhibition of autophagy upon triazine PIP5K inhibitors treatment through GFP-RFP-LC3B baculoviral transfection

HepG2 cells were cultured on coverslips and transfected with a baculoviral vector containing acid-sensitive-GFP and acid-insensitive-RFP LC3B sequences (Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit) according to the manufacturer's instructions. The transfected cells were starved by replacing the complete media with plain media and treated with NG-TZ-17 and IITZ01. Transfected cells were treated with inhibitors for 24 h, fixed with 4% PFA for 20 min, and mounted on slides with DAPI-gold antifade. Images were captured using a Leica confocal microscope with 63X objective magnification. Individual GFP and RFP LC3B puncta were counted and plotted in a graph to indicate the shift in expression following treatment. The effect of treatment on PIP5K1B and lysosomes, analyzed using dual lysotracker and PIP5K immunostaining, is described in the supplementary file.

Effect of PIP5K inhibition on ROS mediated apoptosis

Furthermore, we investigated the effect of the newly synthesized IITZ01 derivatives on ROS-mediated apoptosis. Further, to explore whether treatment with PIP5K inhibitors sensitized HepG2 cells to mild concentrations of H₂O₂ (12.5 µM), HepG2 cells were exposed to a mild concentration of H₂O₂ and treated with two concentrations [IC25 (L) and IC50 (H)] of investigational NG-TZ-17 (17) and IITZ01 (0.5 and 1µM) NG-TZ-20 (20) (2.83 and 5.66 µM) along with single concentrations of standards 67.10 µM chloroquine (CQ, autophagy inhibitor), 7.76 µM ISA-2011B (PIP5K1A inhibitor), and 5 µM ML-385 (Nrf2 inhibitor) in the presence of a proliferative concentration of H₂O₂ for 24 h. Additionally, the effect of these compounds on mitochondrial superoxide was assessed using MitoSOX dye, and autophagy turnover was assessed using Lysotracker and PIP5K1B dual staining. The impact on PIP5K1A, PIP5K1B, Beclin-1, Nrf2, Bax, and BCl-2 levels was assessed using western blot analysis, as described in the supplementary file.

In vivo efficacy of PIP5K inhibitors in HepG2 cells induced tumor model

HepG2/GFP (1X10⁷) cells were dispersed in a 50 µl mixture of MEM and ECM gel matrix and injected subcutaneously into 4-5weeks old SCID mice (Vivo Bio Tech Ltd, Telangana, India). The animals were maintained in individually ventilated cages at 25 ± 5°C with a relative humidity of 50 ± 10% under a pathogen-free environment. All experiments were performed in accordance with the Institutional Animal Ethics Committee (IAEC-IICT-Hyderabad, India). After the tumors reached 200 mm³, the animals were randomly divided into different experimental groups (n = 4). NG-TZ-17 and IITZ01 were dissolved in a vehicle consisting of 5% DMSO + 10%PEG + 10%Tween 80 + 75% ddH₂O. Standard sorafenib was dissolved in 30% capsitol in water and orally administered every day for 10 days. NG-TZ-17 and IITZ01 were administered at 50 mg/kg and standard Sorafenib was administered at 60 mg/kg. The tumor volumes and animal weights were recorded by two independent observers who were blinded to the experimental groups. Tumor volumes were calculated using the formula TV = (l × b²)/2, where l is the tumor length and b is the tumor width. In vivo GFP intensities of tumors were recorded through the IVIS spectrum in the vivo imaging system (Perkin Elmer, San Diego, CA, US). After the experimental period, tumors were collected, weighed, and processed to explore changes in protein expression by extracting proteins using Tissue Protein Extraction Reagent (T-PER) buffer, followed by western blotting.

Statistical analysis

All data are represented as the mean ± SEM, and the significance of variation between the groups was determined by unpaired Student's t-test or one-way analysis of variance followed by Bonferroni's multiple comparison tests using GraphPad Prism, version 8.0. Differences were considered statistically significant at P < 0.05. All experiments were carried out in triplicate unless mentioned otherwise.

PIP5K expression increased along with autophagy and antioxidant markers in HCC patient samples and cell lines

To investigate the potential link between the PIP5K-autophagy and Nrf2 and to explore the role of PIP5K in the progression of hepatic cancer, we estimated the expression of PIP5K1A, PIP5K1B, Beclin-1, and Nrf2 in 36 human HCC patient samples. We compared the expression of these markers in various cancer grades (G1-G3) with that in normal adjacent control tissues (UAT). We observed a grade-dependent increase in PIP5K1A, PIP5K1B, Beclin-1, and Nrf2 expression. PIP5K1A and PIP5K1B expression levels were elevated in all three grades of cancer (G1, G3 * (p < 0.05), and G2 ** (p < 0.01)). We observed the highest expression of the PIP5K isoforms in G2. A similar trend was observed for Nrf2 expression (G1 * (p < 0.05) and G2 **(p < 0.01)). However, in the case of autophagy marker Beclin-1, we observed a steady grade-dependent increase in expression, with the highest expression observed in G3 (**, p < 0.01), when compared to normal adjacent control tissues (Fig. 1A, B, C, and D).

A similar trend of positive correlation between PIP5K isoforms, Beclin-1, and Nrf2 was observed when we estimated the basal expression levels in various HCC cell lines such as PRF5, SNU387, Skhep-1, and HepG2 (Fig. 1E and F). The expression levels of PIP5K isoforms, Beclin-1, and Nrf2 were low in PRF5 and SNU-387 cell lines, whereas the expression of PIP5K isoforms (PIP5K1A and B) was higher in HepG2 cells (** p < 0.01 for both PIP5K1A and B) compared to PRF5 cells. Correspondingly, Beclin-1 and Nrf2 (* p < 0.05, Beclin-1 and *** p < 0.001 Nrf2 compared to PRF5) levels were elevated in HepG2 cells. Keap-1 is a degrading protein responsible for preventing nuclear translocation of Nrf2. Basal Keap-1 levels were low in HepG2 cells and highest in Skhep-1 (*** p < 0.001 compared to PRF5). The Nrf2 dependant-antioxidant enzymes HO-1 (** p < 0.01), SOD2 (* p < 0.05), and AKR1B10 (*** p < 0.001) were also elevated in HepG2 cells compared to the PRF5 cell line (Fig. 1C and D). The positive correlation between proliferative markers (PIP5K isoforms) and adaptive markers (such as Beclin-1, Nrf2, HO-1, and SOD2) suggests that these markers work together to sustain proliferation and energy supply and counter ROS. Because HepG2 cells express high basal levels of PIP5K isoforms, autophagy, and Nrf2-mediated antioxidant markers, HepG2 cells were selected for further experiments.

PIP5K resists ROS-mediated autophagic cell death

ROS govern various cell phases such as proliferation, adaptation, and death by modulating the expression levels of lipid kinases. Thus, to establish the role of ROS in the modulation of PIP5K and the autophagy-Nrf2 adaptive axis, we treated HepG2 cells with increasing concentrations of H₂O₂ (6.25, 12.5, 25, 50, 100, and 200 µM) and explored its effect on cell viability, mitochondrial superoxide (ROS), lysosomal turnover (autophagy flux), expression of PI3K-Akt, autophagy, antioxidant, and apoptosis markers (Fig. 2). To estimate the concentrations of H₂O₂ (ROS) responsible for proliferation, stasis, and death, HepG2 cells were treated with increasing concentrations of H₂O₂ and subjected to the MTT assay. We observed a dose-dependent increase in cell proliferation at lower doses of 6.25 (###, p < 0.001 vs. untreated cells) and 12.5 µM (##, p < 0.01, vs. untreated cells). Reactive Oxygen Species (ROS) generated aided growth as an autophagy-mediated antioxidant pathway countered the ROS, and the energy generated in turn drove the cell growth observed in increased cell proliferation at lower H₂O₂ concentrations. This was followed by an adaptive phase, as observed at 25 and 50 µM, where cells that underwent autophagy induction could not generate sufficient energy and nutrients for cell division, as indicated by reduced cellular proliferation, high ROS levels, and lysosome turnover. This phase was followed by autophagic cell death at higher H₂O₂ concentrations (100 and 200 µM), where elevated levels of ROS triggered autophagic cell death. Such dose-dependent behavior with increasing H₂O₂ concentration was not observed in the serum-containing complete media (Fig. 2A). The MitoSOX assay showed that mitochondrial superoxide concentration following H₂O₂ exposure increased with increasing H₂O₂ concentration. Serum starvation increased ROS levels compared to cells grown in complete media [*, p < 0.05, compared to complete media (CM)]. ROS levels remained fairly unchanged even at increased H₂O₂ concentrations (50 µM). However, at cytotoxic concentrations of 100 and 200 µM, mitochondrial superoxide levels further increased compared to those in the starvation control. The increase in mitochondrial superoxide concentration was ## p < 0.01 and ### p < 0.001, respectively, when compared to the serum starvation control (Fig. 2C). Concurrently, when we measured lysosomal turnover indicative of autophagic flux increased following starvation (*, p < 0.05, compared to CM) following exposure to H₂O₂ in serum-starved cells, we observed a dose-dependent increase in lysosomal turnover compared to CM (Fig. 2B). H₂O₂ at 200 µM induced more autophagic-lysosomal turnover than serum starvation (###, p < 0.001 compared to serum starvation), resulting in autophagic cell death (Fig. 2B). Taken together, the results of cellular proliferation, MitoSOX, lysotracker staining, and mitochondrial superoxide (ROS) indicated that autophagy was activated. The elevated autophagy resisted an increase in mitochondrial superoxide at mild concentrations (6.25 and 12.5 µM), which induced cellular proliferation at moderate concentrations (25 and 50 µM) and promoted cellular stasis. However, cytotoxic concentrations of 100 and 200 µM enhanced ROS to cytotoxic levels, thereby overactivating autophagy and leading to autophagic cell death (Fig. 2B). Dual PIP5K1B and lysotracker staining indicated that both PIP5K1B expression and lysosomal turnover were elevated by 12.5 µM H₂O₂ (Fig. 2D). Western blotting showed that gradient-increasing H₂O₂ exposure affected the expression of PIP5K isoforms (PIP5K1A and B), Nrf2, SOD2, and HO-1. Serum starvation alone increased PIP5K1A, PIP5K1B, and SOD2 expression compared with CM (*, p < 0.05, p < 0.01). There was a consistent increase in the expression of PIP5K1A (** p < 0.001 vs. CM and #p < 0.05 vs. starvation), PIP5K1B (**p < 0.01 vs. CM and #p < 0.05 vs. starvation), Beclin-1 (**p < 0.01), and SOD2 (**p < 0.01 vs. CM and #p < 0.05 vs. starvation) as the concentration of H₂O₂ increased to cytostatic levels at 50 µM (Fig. 2E and F). However, Nrf2, HO-1, Bax, and BCl2 expression remain unaffected by treatment with 50 µM H₂O₂. However, at a cytotoxic concentration, especially at 200 µM, the expression of PIP5K1A, PIP5K1B, Nrf2 (## p < 0.01 vs. starvation), SOD2 (# p < 0.01 vs. starvation), HO-1 (###, p < 0.001 vs. starvation), and Beclin-1 (**, p < 0.001 vs. CM and #, p < 0.05, vs. starvation) were decreased. SRC (##, p < 0.001 vs. starvation), a marker responsible for the relocation of PIP5K from membrane-based signaling to cytosolic and apoptotic proteins Bax/BCl2 ratio increased following 200 µM H₂O₂ exposure (Fig. 2D and E). These results indicate that cells increased PIP5K expression to maintain both PI3K-Akt proliferative and autophagy signaling, upregulating Nrf2 mediated antioxidant defence markers, and increasing SRC indicated a shift from membrane-based proliferative PI3K-Akt to cytosolic autophagy signaling (9, 10). However, at a cytotoxic concentration of 200 µM, the expression of PIP5K isoforms Nrf2, SOD2, and HO-1 decreased, while that of SRC and Beclin-1 increased. This indicates that cells exposed to high H₂O₂ (ROS) concentrations could not modulate ROS even by upregulating autophagy and the Nrf2 mediated antioxidant defence pathway, thus resulting in the overactivation of autophagy, causing autophagic cell death (Fig. 2D and E). Thus, to mimic tumor microenvironment, with elevated ROS, limited nutrients, and cells in a rapidly proliferative phase, HepG2 cells exposed to mild ROS (12.5 µM H₂O₂) along with serum-starvation condition was chosen as it exhibited consistent results with increased cellular proliferation, autophagy, and antioxidant defence mechanism.

Identification of new lead PIP5K inhibitor NG-TZ-17

Under lead optimization, biologically active derivatives of IITZ01 were screened for PIP5K1B inhibition, and NG-TZ-17 and NG-TZ-20 also exhibited PIP5K1B inhibition of 73% and 57%, respectively, at 1 µM (Fig. 3A). The PIP5K1B enzyme inhibition IC50 values of the identified inhibitors NG-TZ-17, IITZ01, and NG-TZ-20 were 239, 281, and 691 nM, respectively (Fig. 3B). Structure-activity relationships revealed that compounds substituted with phenyl groups (IITZ01, NG-TZ-20) showed decreased activity compared to the simple phenyl group (NG-TZ-17).

Triazine PIP5K inhibitors were superior to standard PIP5K1A and autophagy inhibitors in inhibiting proliferation and autophagy

We explored the efficacy of investigational triazine PIP5K inhibitors IITZ01, NG-TZ-17, and NG-TZ-20, along with standards PIP5K1A and autophagy inhibitors ISA-2011B and Chloroquine (CQ) in hepatic cancer cell lines such as HepG2, SNU-387, Skhep-1, and PRF5 (Fig. 3C). We observed that IC₅₀ of NG-TZ-17, IITZ01, NG-TZ-20, ISA-2011B and chloroquine (CQ) in Hepatic cancer were 0.94, 1.63, 5.66, 7.76, and 67.10 µM, respectively in HepG2. IC₅₀ of NG-TZ-17, IITZ01, NG-TZ-20, ISA-2011B and chloroquine (CQ) were 6.48, 1.87, 2.59, 5.59, and 20.69 µM, respectively in SNU-387 cell line. In Skep-1, the concentrations of NG-TZ-17, IITZ01, NG-TZ-20, ISA-2011B, and chloroquine (CQ) they were 2.54, 3.21, 6.47, 8.20, and 54.75 µM, respectively. Finally, in PRF5, IC₅₀ of NG-TZ-17, IITZ01, NG-TZ-20, ISA-2011B, and chloroquine (CQ) were 1.70, 2.80, 4.07, 4.21, and 11.13 µM, respectively. Overall, we observed that the triazine PIP5K1B inhibitors NG-TZ-17 and IITZ01 showed better inhibition of proliferation than standard ISA-2011B (PIP5K1A inhibitor) and chloroquine (autophagy inhibitor). NG-TZ-20 proliferation inhibitory activity was equipotent to that of standard ISA-2011B and superior to that of chloroquine (CQ) (Fig. 3C). In order to explore the effect of triazine PIP5K inhibitors on autophagy, we treated HepG2 cells with two doses of NG-TZ-17 [0.5 µM (17 L) and 1 µM (17H)], IITZ01 [0.5 µM (IITZ01 L) and 1 µM (IITZ01 H)], and NG-TZ-20 [(2.83 µM (20 L) and 5.66 µM (20H)]. Acridine orange-red staining indicated that NG-TZ-17, IITZ01, and NG-TZ-20 deacidified acidic vesicles inhibited autophagy. The treatment resulted in a decrease in acidic vesicles NG-TZ-17 (##, p < 0.01, and ###, 0.001 for NG-TZ-17 at low and high concentrations, respectively, vs. starvation control), and both concentrations of NG-TZ-20 and IITZ01 significantly reduced acidic vesicles (###, p < 0.001 vs. starvation control) (Fig. 3D). To determine the stage-specific inhibition of autophagy, we transfected HepG2 cells with the LC3B-GFP-RFP peptide, which is incorporated into autophagosomes when autophagy is stimulated. Treatment with the triazine PIP5K inhibitors NG-TZ-17 and IITZ01 resulted in an increase in both GFP-LC3B and RFP-LC3B puncta, indicating inhibition of autophagy at the autolysosomal step (Fig. 3E and F). Furthermore, we performed PIP5K1B immunostaining and lysotracker dual staining (Fig. 4). PIP5K inhibitors reduced PIP5K1B expression and lysosomes at high doses of NG-TZ-17, IITZ01, and ISA-2011B. The chloroquine (CQ) standard successfully reduced lysosomal expression (Fig. 4). However, chloroquine treatment did not affect the expression of PIP5K1B. These results indicate that the inhibition of autophagy following treatment with triazine derivatives was due to a decrease in PIP5K1B expression.

Treatment with PIP5K inhibitor sensitized HepG2 to the mild concentration of H₂O₂

Tumor cells modulate ROS levels to maintain high proliferation (11). Tumor cells modulate the ROS switch between proliferation and adaptive pathways (11). Thus, to mimic the tumor microenvironment with elevated ROS levels, we stimulated HepG2 cells with a low concentration of H₂O₂. We exposed the starved HepG2 cells to 12.5 µM H₂O₂. The H₂O₂ exposed cells were then treated with two doses of NG-TZ-17 [(0.47 (17 L) and 0.9 µM (17H)] and IITZ01 [0.5 (IITZ01 L) and 1 µM (IITZ01 H)] along with standards 7.76 µM of ISA-2011B (PIP5K1A inhibitor), 67.10 µM of Chloroquine (autophagy inhibitor), and 5 µM ML-385 (Nrf2 inhibitor). We estimated the levels of mitochondrial superoxide, PIP5K1B, lysosomal turnover, and expression of PI3K-Akt, autophagy, and Nrf2 antioxidant defense markers following treatment with inhibitors.

The MitoSOX staining results revealed that mitochondrial superoxide levels were increased when stimulated with 12.5 µM of H₂O₂ (*, p < 0.05 vs. CM). Treatment with PIP5K inhibitors NG-TZ-17, IITZ01, and ISA-2011B, along with autophagy (CQ) and Nrf2 (ML-385) inhibitors, increased mitochondrial superoxide levels (Fig. 5A and B). The effects of PIP5K inhibitor treatment were similar to those of Nrf2 (ML-385) and autophagy inhibitors (chloroquine). High doses of NG-TZ-20 and IITZ01 significantly increased mitochondrial superoxide levels (#, p < 0.05) compared to starved cells exposed to H₂O₂ (12.5µM) (Fig. 5A and B). Further, to determine the molecular effects of treatment with PIP5K inhibitor on H₂O₂ exposed cells, we explored the effects of treatment on the PI3K-Akt, autophagy, and Nrf2 pathways. H₂O₂ (12.5 µM) exposure increased p-mTOR (**, p < 0.01), Nrf2 (*, p < 0.05), PIP5K1B (***, p < 0.001), PIP5K1A (***, p < 0.001), Beclin-1 (*, p < 0.05), and SRC (*, p < 0.05) compared to cells grown in complete media (Fig. 4E and F). Treatment resulted in decrease in mTOR (#, p < 0.05, for both concentrations of IITZ01 H and ISA-2011B), Nrf2 (#, p < 0.05for17 H, IITZ01 H, ISA-2011B, CQ, and ###, p < 0.001 for ML-385), PIP5K1B (###, p < 0.001 for 17H and ##, p < 0.01 for both concentrations ofIITZ01), PIP5K1A (##, p < 0.01 for 17H, #, p < 0.05 for both concentrations of IITZ01, and ###, p < 0.001 for ISA-2011B), p-AKT (#, p < 0.05 for 17H, both concentrations of IITZ01 and ISA-2011B), HO-1 (#, p < 0.05 for 17H, and both concentrations of IITZ01), SOD2 (#, p < 0.05 for 17H, for both concentrations of IITZ01, ISA-2011B, and CQ), Beclin-1 (#, p < 0.5 for 17H and IITZ01 L), Bax/BCL2 ratio(##, p < 0.01 for 17 L, ###, p < 0.001, for 17H and #, p < 0.05 for IITZ01 H) and no significant changes in SRC expression levels compared to 12.5 µM H₂O₂ exposed group (Fig. 5B and C). Furthermore, to validate the results of molecular analysis, we performed PIP5K1B and lysotracker dual staining, where we observed that Chloroquine, and ML-385 did not have any effect on PIP5K1B expression, although chloroquine was able to inhibit autophagy, as indicated by an increase in Lysotracker fluorescence. Treatment with PIP5K inhibitors, both investigational NG-TZ-17, NG-TZ-20, and IITZ01, and standard ISA-2011B, resulted in a decrease in PIP5K1B and lysosome expression (Fig. 6). These results indicate that NG-TZ-17, IITZ01, and ISA-2011B (PIP5K inhibitors) are superior to the standard autophagy (chloroquine (CQ)) and Nrf2 (ML-385) inhibitors. Furthermore, the triazine lysosomotropic PIP5K inhibitors NG-TZ-17 and IITZ01 were superior to the standard ISA-2011B (PIP5K1A) inhibitor in sensitizing HepG2 cells to a mild concentration of H₂O₂. This was due to the dual inhibition of the PI3K-AKT and autophagy pathways.

Inhibition of proliferative, autophagy and Nrf2 pathways by PIP5K inhibitors reduced tumor burden in HepG2-induced hepatic cancer in SCID mice

GFP-tagged HepG2 cells were injected subcutaneously into the flank region of SCID mice. After the tumors attained 200 mm³volume, mice were treated with PIP5K inhibitors (NG-TZ-17 and IITZ01) and sorafenib for 7 days (Fig. 7A). Tumor-induced mice were treated with dailyNG-TZ-17 (50 mg/kg), IITZ01 (50 mg/kg), or sorafenib (60 mg/kg) for 10 days. The treatment resulted in a decrease in tumor burden, weight, and volume (Fig. 7B-E). The reduction in tumor burden was equipotent to sorafenib (***, p < 0.001 for NG-TZ-17, IITZ01, and Sorafenib when compared to tumor-bearing mice) (Fig. 7E). Treatment with PIP5K inhibitors and sorafenib increased the overall survival of the tumor-induced mice compared to that of the control tumors (Fig. 7C). Western blot examination of isolated liver carcinoma tumors revealed that treatment with PIP5K inhibitors (NG-TZ-17 and IITZ01) reduced the proliferative pathway, as evidenced by a decrease in PIP5K isoforms such as PIP5K1A (**, p < 0.01 for NG-TZ-17, IITZ01, and * p < 0.05, for sorafenib), PIP5K1B (***, p < 0.001 for NG-TZ-17 and IITZ01), and p-Akt (*, p < 0.05 for NG-TZ-17, Sorafenib and **, p < 0.01 for IITZ01). PIP5K treatment also inhibited autophagy, as indicated by a reduction in Beclin-1 levels (***, p < 0.001 for NG-TZ-17 and *, p < 0.05 for IITZ01). Inhibition of the proliferative and autophagy pathways resulted in inhibition of Nrf2 dependant antioxidant pathway, as indicated by the depletion Nrf2 expression (**, p < 0.01 for NG-TZ-17 and IITZ01). Treatment with both PIP5K inhibitors (NG-TZ-17 and IITZ01) and sorafenib induced apoptosis, as indicated by an increase in Bax/BCL-2 ratio expression (**, p < 0.01 for NG-TZ-17, IITZ01, and sorafenib), when compared to the tumor-bearing group (Fig. 7F and G).

Hepatocellular carcinoma is challenging to treat because tumor cells are typically surrounded by hepatocytes responsible for drug metabolism and clearance. Many recently approved and latest in-line cancer chemotherapies have mainly focused on inhibiting proliferative pathways. This inadvertently causes autophagy-induced resistance, which ultimately results in treatment failure. The failure of PI3K/Akt/mTOR inhibitors to treat solid tumors is often linked to the activation of autophagy (12). Inhibition of autophagy alone has only cytostatic effects and increases the likelihood of resistance via the Nrf2-mediated antioxidant pathway (4, 14–16). To cure cancer, proliferative and adaptive pathways must be inhibited. (5, 13–15). Hence, there is a need to inhibit both proliferative and adaptive pathways for the effective treatment of cancer.

Phosphatidyl-inositol-4,5-diphosphate (PI4,5P2), a secondary messenger generated by PIP5K, is employed as a substrate for PI3K signaling and recruits clathrins to recycle lysosomes from autolysosomes during late autophagic events. (16, 17). In mammals, six genes encode kinases involved in the generation of PI4,5P₂. PIP5K1A, PIP5K1B, and PIP5K1C generate PI4,5P₂ from phosphatidylinositol-4-phosphate (PI4P), and PIP4K2A, PIP4K2B, and PIP42C generate PI4,5P₂ from phosphatidylinositol-5-phosphate (PI5P). The availability of PI4P was > 100 times more than PI5P. This suggests greater integration of PIP5K into cell signaling involved in tumor initiation and progression (18). Thus, targeting PIP5K will result in a multifaceted response to inhibit cancer proliferation and adaptation. The PIP5K1A and PIP5K1B isoforms mediate the PI3K-Akt and autophagy pathways, while PIP5K1C controls Rho/Rac1 signaling for cytoskeletal remodelling and cell motility (19).

Singhal et al. (1994) found that PIP5K is essential for hepatic cancer progression and can be an attractive target for its treatment (20). Later, the upregulated PIP5K also contributed to increased tumor aggressiveness, invasion, and metastasis by manipulating filamin and cell adhesion proteins in patients with hepatocellular carcinoma (21). Further studies have been conducted by Semenas et al. (2014), Sarwar et al. (2019), and Peng. J et al. 2021, demonstrated a positive correlation between PIP5K1A and PIP5K1C expression as prostrate and hepatocellular carcinoma progression (16, 21). To date, there have been no reports available on PIP5K1B contribution to hepatic cancer progression. Understanding the relevance of PIP5K in anticancer treatment, Semenas et al. 2014, and Martuza Sarwar identified ISA-2011B as a small molecule PIP5K1A inhibitor and explored its efficacy in the treatment of prostate and breast cancer via PI3K-Akt inhibition. (16, 21, 22). Numerous studies have shown that ROS regulate lipid kinases to modulate the proliferative (PI3K-Akt) and adaptive (Autophagy) pathways, affecting cellular proliferation, stasis, and death (23, 24). These studies did not explore how reactive oxygen species modify PIP5K levels or how changing PIP5K isoforms, autophagy, and antioxidant indicators contribute to liver cancer progression and resistance. Since PIP5K1A and PIP5K1B are key isoforms in PI3K-Akt and autophagy signaling, this study used isoform-specific PIP5K inhibitors to determine their roles in ROS-assisted HCC progression.

In the present study, we estimated PIP5K isoforms, autophagy, and Nrf2 expression in 36 progressive grade hepatic cancer patients and compared them with normal adjacent controls. We observed that the expression of PIP5K isoforms Beclin-1 and Nrf2 increased as the grade progressed, indicating a positive correlation between the markers (Fig. 1). Our results are consistent with those reported by Semenas et al. (2014) and Peng. J et al. 2021, observed an increase in PIP5K1A and PIP5K1C levels as prostate cancer and HCC progressed (16, 21). In addition, Beclin-1 and Nrf2 must be upregulated to counteract reactive and restore the energy and nutrient supply to sustain proliferation. Studies have also linked Nrf2 levels to hepatocellular carcinoma progression (25, 26). These investigations revealed a link between PIP5K, autophagy, and antioxidant defense pathways, which needs further study to become a target. Our estimates of PIP5K isoforms, autophagy markers, and Nrf2 baseline expression levels in hepatic cancer cell lines (PRF5, SNU-387, Skhep-1, and HepG2) showed a similar pattern. PIP5K isoforms elevated autophagy and Nrf2-mediated antioxidant enzymes; HepG2 cells had the highest expression levels of PIP5K isoforms, autophagy, and Nrf2-mediated antioxidant markers. Thus, HepG2 cells were selected for further experiments. Next, we exposed HepG2 cells to serially increasing H₂O₂ concentrations related to the effects of ROS produced by autophagic flux, proliferation, autophagy, and Nrf2 pathways (Fig. 2). We observed that in the presence of serum starvation, lower H₂O₂ concentrations (6.25 and 12.5 µM) induced proliferation, while moderate concentrations (25 and 50 µM) showed a stasis effect, where neither proliferation nor death was observed; however, higher H₂O₂ concentrations (> 100 µM) resulted in a significant loss of cell viability (27). A similar trend was not observed in HepG2 cells treated with complete media. This explains why cells maintain a higher proliferative state even with scarce nutrient supply (28, 29). ROS and autophagy levels increased with H₂O₂ concentration and were the highest at cytotoxic doses, MitoSOX, and lysotracker staining. This suggests that progressively increasing H₂O₂ concentrations produced high ROS levels in the form of mitochondrial superoxides, causing autophagic cell death.

To understand the relationship between PI5K isoforms and proliferation, autophagy, and antioxidant defense mechanisms as ROS levels progressively increase, we estimated the levels of PIP5K1A, PIP5K1B, SRC, Beclin-1, Nrf2, SOD2, HO-1, Bax, and BCl2 to understand how ROS modulate proliferation, autophagy, and antioxidant defense mechanisms. We observed that as ROS levels progressively increased, PIP5K expression was induced in HepG2 cells. Elevated levels of both proliferative and autophagy proteins upregulated Nrf2 (nuclear transcriptional regulator of antioxidant enzymes) production by activating Antioxidant-Response-Elements (DNA sequences encoding for antioxidant enzymes) and aided in nuclear translocation, thereby increasing the expression of Nrf2-mediated antioxidant genes. The high levels of the PI3K-Akt pathway synthesize Nrf2, and activated autophagy aids in Nrf2 nuclear translocation by degrading Keap-1 (a protein adaptor unit complexes with Nrf2 for its degradation), thus increasing antioxidant enzymes such as HO-1 and SOD1 (30, 31). However, at cytotoxic levels, as shown in previous studies, the expression of PIP5K isoforms and proliferative markers declines as the SRC kinase-mediated feedback pathway is activated, indicating a shift from a proliferative state to stasis and death by maintaining elevated autophagy levels (9, 32, 33). Our results correlate with those of Halstead et al. (2006), who showed that depletion of PI4,5P₂ is crucial for oxidative stress-induced apoptosis. The study also demonstrated that the overexpression of PIP5K rescued cells from H₂O₂-induced apoptosis (33).

The available PIP5K isoforms are utilized to maintain elevated autophagy levels, which further result in autophagic cell death by activating apoptosis (17, 33). We identified our in-house molecule, IITZ01 (an autophagy inhibitor), and its derivatives inhibited PIP5K through cell-free kinase inhibition assays. The IC50 values of the identified inhibitors NG-TZ-17, IITZ01, and NG-TZ-20 were 239, 281, and 691 nM against PIP5K1B enzyme, respectively. Our inhibitor retained autophagy inhibition characteristics while inhibiting PIP5K1B, as indicated by acridine orange staining, GFP-RFP-LC3B baculoviral transfection, and dual PIP5K1B and lysotracker staining (Figs. 3 and 4).

Since ROS modulate PIP5K expression and cellular proliferation, treatment with PIP5K pharmacological inhibitors at a proliferative concentration of H₂O₂ should sensitize cells to apoptosis. Thus, we exposed cells were to mild ROS (12.5 µM H₂O₂) following serum starvation and treated cells with increasing two concentrations of lysosomotropic PIP5K inhibitors like NG-TZ-17 and IITZ01, and compared its results to standards PIP5K1A (ISA-2011B), autophagy (CQ) and Nrf2 (ML385) inhibitors. We observed that lysosomotropic PIP5K inhibitors inhibited both the proliferative PI3K-Akt and autophagy pathways, which decreased the expression of Nrf2 and other Nrf2-mediated antioxidant genes, compared to standard PIP5K (ISA-2011B), autophagy (CQ), and Nrf2 (ML385) inhibitors. This is possibly due to dual inhibition of proliferative (PI3K-Akt) and adaptive (Autophagy) pathways, which ultimately decreases the expression of Nrf2 and Nrf2-mediated antioxidant genes, resulting in sensitization of HepG2 cells to apoptosis in a proliferative concentration of H₂O₂ as previously explained (Figs. 5 and 6). PIP5K inhibitors overcame the limitations of individual autophagy (CQ) and Nrf2 (ML385) inhibitors.

Treatment with PIP5K inhibitors NG-TZ-17 and IITZ01 at 50 mg/kg orally administered daily for 7 days reduced tumor burden and tumor mass, and improved overall survival of HepG2-induced hepatic cancer in SCID mice. The overall in vivo efficacy of the PIP5K inhibitors was similar to that of standard sorafenib 60 mg/kg orally administered daily for a similar 7 days period. However, molecular analysis of isolated tumors revealed that PIP5K inhibitor treatment offers the advantage of inhibition of resistance-inducing autophagy and Nrf2 pathways. The treatment with PIP5K resulted in dual inhibition of proliferative and adaptive (autophagy and Nrf2) pathways as indicated by decrease in PIP5K isoforms (PIP5K1A and PIP5K1B), Akt, Beclin-1, Nrf2 compared to only PIP5K1A and Akt inhibition in the case of Sorafenib treatment. However, treatment with both PIP5K inhibitors (NG-TZ-17 and IITZ01)/ sorafenib induced apoptosis, as indicated by an increase in the Bax/BCL-2 ratio (Fig. 7). Although inhibition of the proliferative pathway by sorafenib treatment is sufficient to induce apoptosis and reduce tumor burden, it had minimal effect on resistance-inducing autophagy and the Nrf2 pathways responsible for its treatment failure (34, 35). These results demonstrate the advantage of using PIP5K inhibitors over sorafenib for HCC treatment, as they inhibit both the proliferative and adaptive pathways.

Overall, this study demonstrates that PIP5K mediates cellular proliferation and adaptation against excess reactive oxygen, and that inhibition of PIP5K is crucial for sensitizing cancer cells to even minute amounts of reactive oxygen species. Although both PIP5K1A and PIP5K1B are involved in PI3K-Akt and autophagy signaling, based on in vitro results, triazine PIP5K inhibitors were effective in inhibiting the proliferative (PI3K-Akt) and autophagy pathways as compared to standard ISA-2011B (PIP5K1A inhibitor). This could be due to the drug-like properties of triazine PIP5K inhibitors. Further detailed comparative studies are required to explore the drug-like properties of PIP5K inhibitors. Targeting PIP5K becomes even more crucial and attractive as some Akt mutations in cancer utilize PIP₂ as a substrate instead of PIP₃. The effect of PIP5K inhibition on cancer cell glycolysis, the insulin pathway, and the aldose reductase-mediated Warburg effect must be studied to understand the full impact on PIP5K inhibition.

The study shows that Cancer cells regulate ROS levels through the PIP5K-Autophagy-Nrf2 axis. ROS modulation helps cancer cells survive nutritional deprivation and proliferate. In vitro, the PIP5K inhibitors NG-TZ-17, IITZ01, and ISA-2011B sensitized HCC cells to moderate ROS levels. In vivo, the PIP5K inhibitors NG-TZ-17 and IITZ01 decreased tumor burden in HCC-induced SCID mice and were comparable to sorafenib. Due to its simultaneous PI3K-Akt and autophagy suppression, PIP5K inhibitors overcame the limitations of sorafenib.

PIP5K (Phosphatidyl-inositol-4-Phosphate-5-Kinase), PI3K (Phosphoinositide 3-kinases), mTOR (Mammalian target of rapamycin), ROS (Reactive Oxygen Species), Nrf2 (nuclear factor erythroid 2–related factor 2), HCC (Hepatocellular Carcinoma), IHC (Immunohistochemistry), IF (Immunofluorescence), GFP (Green Fluorescent Protein), RFP (Red Fluorescent Protein), HO-1 (Heme oxygenase-1), SOD2 (superoxide dismutase 2), p.o (per os (oral route)), AALD (Alcohol-Associated Liver Disease), NAFLD (Non-Alcoholic Fatty Liver Disease), VEGF (Vascular endothelial growth factor), p-Akt (Phosphorylated-Akt), AKR1B10 (Aldo-keto reductase family 1 member B10), ATP (adenosine triphosphate), ADP (adenosine diphosphate), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), CQ (Chloroquine), HBSS (Hank's balanced salt solution), SDS-PAGE (Sodium dodecyl-sulfate polyacrylamide gel electrophoresis), ECL (enhanced chemiluminescence), PI4,5P2 (Phosphatidyl-inositol-4,5-diphosphate), PI4P (phosphatidylinositol-4-phosphate)

Acknowledgements

The authors are thankful to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India, and Director of NIPER-Guwahati for providing fellowships and research facilities to the authors. We also thank the Department of Biotechnology (DBT) under the Ministry of Science and Technology, Government of India (BT/PR25319/NER/95/1133/2017) for funding this project. The authors wish to express their sincere thanks for the support and cooperation provided by the DBT-National Liver Disease Biobank for providing the tissue microarray slides for IHC.

Conflict of interest: The authors declare that they have no conflict of interest.

Author contributions: Conceptualization and design, V.G.M. Naidu and Sai Balaji Andugulapati; data curation and analysis, P.A. Shantanu, Vishal Rajdev, and Jagadeesh Kumar Gangasani; writing—original draft preparation, P.A. Shantanu; review and editing, Syamprasad N.P.; Samir Ranjan Panda; S.K. Sarin; Dinesh Mani Tripathi; Sai Balaji Andugulapati; V.G.M. Naidu. All the authors have read and agreed to the published version of the manuscript.

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Targeting Phosphatidyl-inositol-4-Phosphate-5-Kinase ameliorates hepatic cancer by inhibiting PI3K/Akt/mTOR and autophagy mechanism and enhancing ROS-mediated apoptosis

Status:

Version 1

Abstract

Figures

Introduction

Material and Methods

Expression levels of PIP5K and markers of autophagy in Hepatic cancer

Cell culture

Exploring the role of PIP5K in Reactive Oxygen Species (ROS) mediated apoptosis and autophagy

Development of PIP5K inhibitors

Enzyme inhibition

Cell viability studies of PIP5K inhibitors on HCC cell lines

Establishing the phase-specific inhibition of autophagy upon triazine PIP5K inhibitors treatment through GFP-RFP-LC3B baculoviral transfection

Effect of PIP5K inhibition on ROS mediated apoptosis

Statistical analysis

Results

PIP5K resists ROS-mediated autophagic cell death

Identification of new lead PIP5K inhibitor NG-TZ-17

Treatment with PIP5K inhibitor sensitized HepG2 to the mild concentration of H₂O₂

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Targeting Phosphatidyl-inositol-4-Phosphate-5-Kinase ameliorates hepatic cancer by inhibiting PI3K/Akt/mTOR and autophagy mechanism and enhancing ROS-mediated apoptosis

Status:

Version 1

Abstract

Figures

Introduction

Material and Methods

Expression levels of PIP5K and markers of autophagy in Hepatic cancer

Cell culture

Exploring the role of PIP5K in Reactive Oxygen Species (ROS) mediated apoptosis and autophagy

Development of PIP5K inhibitors

Enzyme inhibition

Cell viability studies of PIP5K inhibitors on HCC cell lines

Establishing the phase-specific inhibition of autophagy upon triazine PIP5K inhibitors treatment through GFP-RFP-LC3B baculoviral transfection

Effect of PIP5K inhibition on ROS mediated apoptosis

Statistical analysis

Results

PIP5K resists ROS-mediated autophagic cell death

Identification of new lead PIP5K inhibitor NG-TZ-17

Treatment with PIP5K inhibitor sensitized HepG2 to the mild concentration of H2O2

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Treatment with PIP5K inhibitor sensitized HepG2 to the mild concentration of H₂O₂