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Canagliflozin, a SGLT-2 inhibitor, relieves ER stress, modulates autophagy and induces apoptosis in irradiated HepG2 cells: Signal transduction between PI3K/AKT/GSK-3β/mTOR and Wnt/β-catenin pathways; in vitro

1 Department of Radiation Biology, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo, Egypt
2 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, Cairo, Egypt

Date of Submission14-Dec-2019
Date of Decision18-Jan-2020
Date of Acceptance22-Apr-2020
Date of Web Publication23-Jul-2021

Correspondence Address:
Mohamed Khairy Abdel-Rafei,
Department of Radiation Biology, National Centre for Radiation Research and Technology, Atomic Energy Authority, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_963_19

 > Abstract 

Background and Objectives: Metabolic shifting from mitochondrial respiration to glycolysis characterizes malignant cells from its normal counterparts and is attributed to overactivation of oncogenic signaling pathways. Hence, this study intended to investigate the influence of canagliflozin (CAN) and/or γ-irradiation (γ-IR) on HepG2 cell proliferation, crosstalk between phosphatidylinositol 3-kinases (PI3K)/AKT/glycogen synthase kinase-3-β (GSK3-β)/mTOR and Wnt/β-catenin signaling pathways, and their regulation of diverse processes, such as endoplasmic reticulum (ER) stress, autophagy, and apoptosis.
Materials and Methods: HepG2 cells were treated with different doses of CAN and then exposed to different doses of γ-IR to achieve optimization that was based on cytotoxicity and clonogenic assays, respectively. The effects of CAN and/or γ-IR on glycolytic metabolism, cellular bioenergetics, oxidative stress, ER stress and autophagy biomarkers, expression of PI3K/AKT/GSK3-β/mTOR and Wnt/β-Catenin signaling pathways, and apoptotic markers were monitored.
Results: CAN enhanced the antitumor potential of γ-IR as displayed by a significant inhibition of clonogenic survival in HepG2 cells via inhibition of glucose uptake, lactate release, and modulation of ER stress-mediated autophagy; switched it to apoptosis; as well as disabled signaling pathways which contribute to metabolic reprogramming and tumor progression induced by γ-IR that confer radioresistance and treatment failure.
Conclusion: Our study sheds light on the effective combination of CAN and γ-IR in hepatocellular carcinoma treatment and necessitates CAN treatment prior to γ-IR to overcome metabolic reprogramming-associated radioresistance and improve curative outcomes.

Keywords: Canagliflozin, caspase-12, caspase-3, endoplasmic reticulum-stress, hepatocellular carcinoma

How to cite this URL:
Abdel-Rafei MK, Thabet NM, Rashed LA, Moustafa EM. Canagliflozin, a SGLT-2 inhibitor, relieves ER stress, modulates autophagy and induces apoptosis in irradiated HepG2 cells: Signal transduction between PI3K/AKT/GSK-3β/mTOR and Wnt/β-catenin pathways; in vitro. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=322165

 > Introduction Top

Hepatocellular carcinoma (HCC), one of the globally most fatal human malignant diseases, is distinguished by complex and heterogeneous factors.[1] The huge diversity of molecular patterns in HCC patients arises from the broad range of etiologies that contribute to tumor development, which represents a major dilemma in successful therapy. Although sorafenib and regorafenib are the currently approved treatments in advanced HCC patients,[2] the actual survival and response rates to these therapies are limited. Hence, novel treatments with better outcome are urgently needed.[3] Unlike normal cells, carcinogenic metabolism is thought to consuming glucose and glutamine to producing lactate at prodigious rates for highly proliferative cancer cells to satisfy its biosynthetic precursors including lipids, proteins, and nucleic acids.[4] Cells receive a signal from many different growth factor receptors that induce multiple alterations in cellular signaling machinery, which regulate diverse processes, such as protein synthesis and cell growth, motility, and survival.[5] The serine/threonine kinase, AKT, is a prime example of oncogenic signaling kinases, which pushes the high metabolic demands of cancer cells through increase in aerobic glycolysis via upregulation of glucose transporter levels and suppression of fatty acid β-oxidation.[4] Insulin/insulin-like growth factor (IGF) signaling activates AKT through inducing phosphatidylinositol 3-kinases (PI3K). Then, PI3K phosphorylates phosphatidylinositol-4-5-bisphosphate to PIP3. Afterward, PIP3 activates the phosphatidylinositol-dependent protein kinase 1 (PDPK1) and recruits AKT to the plasma membrane where PDPK1 phosphorylates AKT (T308) in the activation loop. The phosphatase and tensin homolog deleted from chromosome ten (PTEN), is a negative regulator of Akt function through dephosphorylation of 3-OH position of PIP3.[6] There are different downstream targets of AKT interconnected to WNT/β-catenin signaling pathway that are important for cell proliferation and differentiation, and its irregular activation is another major cause of cancer progression. One of these targets is glycogen synthase kinase-3-β (GSK-3-β), which has multiple roles ranging from glucose homeostasis to inflammation, and it plays an important role in WNT/β-catenin signaling activation. Another key target of AKT is mTORC1 that responds to the nutrients and conditions which promote cellular growth. It was shown that inhibition of autophagy by mTORC1 was observed to rescue disheveled (Dvl) that leading to activation of the WNT pathway.[4],[6] Indeed, Wnt/β-catenin signaling was shown to contribute to the metabolic reprogramming and promoted aerobic glycolysis in cancer cells through inhibiting mitochondrial respiration, stimulating the pyruvate carboxylase activity, and enhancing pyruvate dehydrogenase (PDH) kinase-1 expression.[7] Therefore, blockade of PI3K/Akt/GSK-3β and Wnt/β-catenin signaling axis is of deem importance in strategies targeting tumor glycolytic metabolism. Concomitantly with Wnt/β-catenin signaling activation, HCC cells were found to overexpress glucose transporters (GLUT 1 and 3).[3]

In cancer cells, endoplasmic reticulum (ER stress) may restore homeostasis and make the adjacent environment hospitable for cancer survival and cancer expansion. There are three ER stress signaling branches, inositol-requiring enzyme-1α (IRE-1α), activating transcription factor 6 (ATF-6), and pancreatic ER kinase-like ER kinase (PERK) localized in the ER, which are involved in cancer progression. It was shown that IRE-1α and its down-signaling, X-box binding protein-1 (XBP-1), contribute to cancer progression. XBP-1 is increased in many human cancers such as breast cancer, HCC, and pancreatic adenocarcinoma. Under various stressful conditions such as nutrient deprivation, hypoxia, pH changes, or poor vascularization represent a growth limit to cancer cells, and thus activate the unfolded protein response (UPR) and produce ER stress. During carcinogenesis, the high proliferation rates of cancer cells require increased activities of ER protein folding, assembly, and transport, which are conditions that can induce ER stress; that response is considered cytoprotective and is involved in cancer growth and adaptation against harsh environments.[8] Therefore, the ER is one site of interaction between environmental signals and a cell's biological function. It also revealed that the ER is tightly linked to autophagy, inflammation, and apoptosis.[9]

Recent studies indicated that canagliflozin (CAN), the sodium-glucose cotransporter-2 inhibitor (SGLT2-I), an antidiabetic drug, may inhibit the growth of pancreatic and colon cancer cells, potentially through the inhibition of SGLT2-mediated glucose uptake.[10] However, little has been reported on the direct antitumor effects of CAN on HCC with the linkage of cancer metabolic reprogramming. Worthwhile, a promising antiproliferative effect was observed in SGLT2-expressing HCC cells after CAN treatment.[10] The main goal of radiation therapy that remains an important component of cancer therapy is to cease cancer cell proliferation and promote its apoptosis. Hence, here, we investigated the anticancer effect of CAN and irradiation (IR) against HCC (HepG2) proliferation through their glycolytic metabolism; the crosstalk between PI3K/AKT/GSK-3-β/mTOR and WNT/β-Catenin signaling pathways; and regulation of diverse processes, such as ER stress (ATF-6, IRE1α, XBP1, XBP1s, and CHOP), autophagy (JNK and Beclin-1), and apoptosis (BCL-2, Caspase-3, and Caspase-12).

 > Materials and Methods Top

Cell culture, treatment, and reagents

HCC (HepG2) cell line (express wild-type p53) was obtained from the Tissue Culture Unit of the Holding Company for Biological Products and Vaccines (VACSERA), Giza, Egypt, which was supplied through the American Type Culture Collection. CAN (Invokana®) was purchased from Janssen Pharmaceuticals, Inc. While other chemicals and reagents were of analytical grade and obtained from Sigma-Aldrich Chemical Co., USA. The antibodies against various biomarkers were obtained from the following sources: anti-t-JNK (CAT# sc-7345), anti- p-JNKThr183/Tyr185 (CAT# sc-293136), anti-beclin-1 (anti-BECN1; CAT # sc-48341), anti-t-PI3K (CAT# sc-1637), anti-p-PI3KTyr508 (CAT# sc-12929), t-Akt (CAT# sc-5298), p-AktThr308 (CAT# sc-135650), t-PTEN (CAT# sc-393186), anti-t-GSK-3-β (CAT#sc-81462), p-GSK-3-βser9 (CAT#sc-11757), and p-PTENSer380 (CAT# sc-377573), which were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA, whereas antibodies against t-mTORC1(CAT# PA1-518), p-mTORC1Ser2481 (CAT # PA5-77981), t-β-catenin (CAT# MA1-301), and p-β-cateninThr41/Ser45 (CAT# PA5-37545) were obtained from Thermo Fisher Scientific, Waltham, MA, USA.

Subculture of cell line

To verify the degree of confluence and to confirm the absence of contaminants (such as bacterial and fungal), the cultures were viewed using an inverted microscope (CKX41; Olympus, Japan). Briefly, cell monolayer using a volume equivalent half of the volume of the culture medium was washed with phosphate-buffered saline (PBS) free of Ca2+/Mg2+. Then, trypsin/EDTA was added on to the washed cell monolayer using 1 ml/25 cm2 of surface area, and the flask was rotated to merge the monolayer with Trypsin/EDTA. Then, the flask was left for 10 min in the incubator. After that, the cells were examined using an inverted microscope to ensure that all the cells were detached and floated.

Culture media

The cells were cultured in a humid environment (at 37°C and 5% CO2 atmosphere) in RPMI-1640 medium supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum (FBS). For experiments investigating the influence of CAN on glycolytic metabolism (glucose uptake and lactate release as well as PDH activity) and associated cellular responses (adenosine triphosphate [ATP] and reactive oxygen species [ROS] production as well as ER stress and autophagy), the cultured cells were transferred to Dulbecco's minimum essential medium eagle (DMEM) supplemented with 10 mM glucose, 1 mM glutamine without pyruvate supplemented with 10% heat-inactivated dialyzed fetal calf serum (56°C, 30 min), 100 U/ml penicillin G, 100 μg/ml streptomycin, and Fungizone (250 μg/l), 4 h before the experiments. In favor of CAN application to the cultured HCC cells, CAN was dissolved in dimethyl sulfoxide (DMSO) at the indicated concentration and added to the culture medium to achieve a final concentration of 0.1%.

Cellular growth and dose–response curve

Under standard growth conditions, exponentially growing HepG2 cells (5 × 103 cells) were collected using 0.25% trypsin-EDTA and seeded in 96-well plate in DMEM medium. Using (3- [4,5-dimethylthiazol-2-yl]-2.5-diphenyl tetrazolium bromide) (MTT) method to verify the viable cells, a fitted dose–response curve was conducted against increasing doses of CAN ranging from 10 to 60 μM for 24 h. The IC50 values were calculated according to the equation for Boltzmann sigmoidal concentration–response curve using the nonlinear regression models, and the x values were transformed to log form (GraphPad Company, San Diego, California). The results reported are means of at least three separate experiments.

Radiation exposure

The cultured HepG2 cells line was irradiated with a Canadian gamma cell-40 (137Cs) at the NCRRT, Cairo, Egypt, at a dose rate of 0.68 Gy/min for a single shot of 2, 4, and 6 Gy.

Clonogenic assay

Clonogenic assay was performed according to the method of Weigel et al.[11] To assess the surviving fraction of HepG2 cells incubated with CAN and/or γ-IR, the cells (3000 cells/ml) were suspended in 20-ml culture medium and treated with vehicle (DMSO) or CAN (30 μM) for 24 h.

After that, γ-IR doses (2, 4, or 6 Gy) were delivered in order to have the following four categories of culture: control, CAN, γ-IR, and CAN + γ-IR. All types of culture were incubated at 37°C in a humidified atmosphere of 5% CO2 for 2 weeks to allow the formation of macroscopic colonies on polymerized bactoagar plates. After the incubation period, the colonies were stained by 0.5% crystal violet in methanol/acetic acid (7:1) and were counted microscopically. The number of colonies containing ≥50 cells was determined. The surviving fraction of HepG2 cells was determined by the clonogenic assay and calculated relative to the nonirradiated control. Experiments were performed in at least triplicate, and the mean and standard error were calculated.

Culture groups

The cultured HepG2 cells were divided into: Group 1 (HepG2): untreated HepG2 cells (0.1% DMSO in culture medium [V/V]) served as negative control, Group 2; (HepG2 + CAN): HepG2 cells treated with CAN (30 μM dissolved in DMSO at a final concentration of 0.1% [W/V]), Group 3 (HepG2+ γ-IR): HepG2 cells exposed to 6 Gy (single shot), and Group 4 (HepG2 + CAN + γ-IR): HepG2 cells treated with CAN and exposed to 6 Gy.

Cell viability and proliferation assay

HepG2 cell viability and proliferation in all groups was determined using the MTT cell proliferation kit (Trevigen Inc., Gaithersburg, MD, USA) as per manufacturer's protocol. Briefly, 5 × 103 cells were seeded in each well of a 96-well plate in the presence or absence of CAN (30 μM) in a final volume of 100 μl of 10% fetal bovine serum (FBS) culture medium and were allowed to attach overnight. A specific volume of MTT reagent (100 μl/well) was added, and the plate was incubated for 12 h to allow for intracellular reduction of the soluble yellow MTT to the insoluble purple formazan dye. To solubilize the formazan dye before measuring the absorbance of each sample, the detergent reagent was introduced into each well and read in an enzyme-linked immunosorbent assay (ELISA) reader at 550–600 nm.[12] Six wells were used for each group. Cell proliferation was assessed as the percentage of cell proliferation compared to the untreated HepG2 as control cells.

Detecting the gene expression of ATF-6, IRE-1α, XBP1u, XBP1s, CHOP, SGLT-2, BCL-2, and p53 using quantitative reverse transcription-polymerase chain reaction

Real-time-polymerase chain reaction (PCR) was carried out using the StepOne Real-time PCR System Instrument (Life Technologies, Thermo Fisher Scientific Inc., USA) with reaction contained 5-μl SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific Inc., USA), 0.3-μl gene-specific forward and reverse primers (10 μM), cDNA, and nuclease-free water. The cells were harvested, and the whole cell RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA). The cDNA was reverse transcribed from 2 μg of the total RNA with oligo (dT) and M-MLV reverse transcriptase (Promega, Madison, WI, USA). The sequences of PCR primer pairs used for each gene ATF-6, IRE-1α, XBP1u, XBP1s, CHOP, SGLT-2, BCL-2, and p53 are shown in [Table 1]. Data were analyzed with the ABI Prism sequence detection system software and quantified using the v1.7 Sequence Detection Software from PE Biosystems (Foster City, CA, USA). The relative expression of the studied genes was calculated using the comparative threshold cycle (2ΔΔCT) method. All values were normalized to the β-actin, which was used as the control housekeeping gene.[13]
Table 1: Primer sequences used in quantitative reverse transcription-polymerase chain reaction

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Western blot analysis

After 24 h treatment with 0.1% DMSO, 30-μM CAN and 6 Gy γ-IR, or combination of both at 37°C, the cells were harvested and washed once with ice-cold PBS, then lysed with the RIPA lysis buffer, followed by a centrifugation at 14,000 ×g at 4°C for 10 min. The supernatants were collected after boiling for 5 min. Protein contents were measured using the BCA Protein Assay kit (Abcam, Cambridge, MA, USA). Proteins (30 μg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). The membranes were blocked in 5% skim milk at 37°C for 1 h, and then incubated with the primary antibodies at 4°C overnight. Thereafter, the membranes were washed with the TBST buffer (containing 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) three times and incubated at 1:1000 dilutions of secondary antibodies at 37°C overnight on a roller shaker at 4°C. Immunoblot was done with the indicated primary antibody followed by the appropriate horseradish peroxidase-conjugated goat immunoglobulin (Amersham. Life Science Inc., USA). Chemiluminescence detection was performed with the Amersham detection kit according to the manufacturer's protocols and exposed to X-ray film. The amount of the studied protein was quantified by densitometric analysis of the auto-radiograms using a scanning laser densitometer (Biomed Instrument Inc., USA). The results were expressed after normalization to β-actin protein expression (as housekeeping protein).[14]

Determination of glucose uptake and lactate release

HepG2 cells were seeded in DMEM with 10% FBS in 6-well plate at the density of 2 × 105 cells/well. On the next day, the culture medium was replaced with a glucose-rich DMEM (10 mM glucose, 1 mM glutamine, without pyruvate, containing 10% dialyzed FBS and 100 IU/mL penicillin/streptomycin) 4 h before incubation for 24 h with the indicated doses of 30 μM and 6 Gy for CAN and γ-IR, respectively. Spent media was collected, centrifuged at 4000 ×g to remove cell debris, and deproteinized by 5% trichloroacetic acid, and the culture supernatant was obtained for the evaluation of glucose uptake and lactate release rates, using colorimetric glucose and Lactate assay kits (BioVision, San Francisco Bay Area, USA) as per manufacturer's instructions. The uptake rate = (glucose content in the control group − glucose content in the treatment group)/glucose content in the control group × 100%. The release rate = (lactate content in the treatment group − lactate content in the control group)/glucose content in the control group × 100% as described by Zhao et al.[15] All measurements were normalized to cell numbers.

Enzyme-linked immunosorbent assays

In the control and treated HepG2 cells, the level of IGF-1 was determined by using a corresponding ELISA kit purchased from BD Biosciences (San Jose, CA, USA) according to the protocol provided by the manufacturer. The enzymatic activity of PDH was estimated using human PDH ELISA kit (Abcam, Cambridge, MA, USA). Caspase-12 activity was measured using the Human Caspase-12 ELISA kit (Cusabio Biotech, China). In addition, Caspase-3 activity and SGLT-2 protein level were determined by using a corresponding ELISA kit purchased from MyBioSource (San Diego, USA) according to the protocol provided by the manufacturer. The samples were determined using ELISA plate reader (Biotek, Winooski, Vermont, USA.).

Determination of hydrogen peroxide (H2O2) and intracellular adenosine triphosphate levels

The concentration of intracellular H2O2 was measured by using colorimetric assay kit from (Sigma-Aldrich, St. Louis, USA). Intracellular ATP level was measured by using Bioluminescence Assay kit HS II (Roche Applied Science, Basel, Switzerland) as per manufacturer's protocol.

Assessment of intracellular Ca2+ [Ca2+]i concentration

The [Ca2+]i level in the treated and control HepG2 cells was determined by Fura-2 AM fluorescent dye according to the method of Vega et al.[16] After 24 h incubation with the tested treatments, the cells were harvested and rinsed with Krebs–Ringer buffer (pH 7.4), containing 137-mM NaCl, 5-mM KCl, 1-mM MgCl2, 1.5-mM CaCl2, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and then incubated with 5-μM Fura-2 AM (AAT Bioquest, CA, USA) for 1 h at 37°C. Afterward, the cells were rinsed twice and re-suspended in Krebs–Ringer buffer and measured for fluorescence (Fs) using a spectrofluorimeter (PerkinElmer, MA, USA). The employed emission and excitation wavelengths were 510 and 340–380 nm, respectively. The maximal and minimal fluorescence (Fmax and Fmin, respectively) were determined in the presence of 0.1% Triton-X and 10-mM (ethylene glycol tetraacetic acid) EGTA, respectively. The concentrations of [Ca2+]i were calculated according to the following equation: [Ca2+]i = Kd (FsFmin)/(FmaxFs), where Kd for Fura-2 AM equals 224 nM.

Statistical analysis

The data were subjected to one-way analysis of variance, followed by Tukey's multiple comparison post hoc test. Kolmogorov–Smirnov and Bartlett's tests were used to evaluate the normality and homology of variance, respectively. Statistical analyses were performed using Prism, version 6 (GraphPad Software, La Jolla, CA, USA). All the tests were two tailed, and P < 0.05 was considered statistically significant.

 > Results Top

Canagliflozin blocks the cellular proliferation and clonogenic survival of HepG2 cancer cells

For determining the effect of CAN on human HepG2 cell viability, we first assessed the cytotoxic effects of CAN using MTT assay. When HepG2 cells were incubated with increasing concentrations of CAN ranging from 10 to 60 μM for 24 h, the cell viability was statistically significantly reduced (P < 0.05) at the least dose (10 μM), reaching a profound decrease (P < 0.001) at the maximum dose (60 μM), demonstrating a remarkable cytotoxicity of CAN at these concentrations. The calculated IC50 of CAN was 30 μM (antilog 1.47) in HepG2 cells, and DMSO was used as vehicle control for CAN [Figure 1]a. To further estimate whether CAN enhances the cellular sensitivity to γ-IR, HepG2 cells were incubated with CAN at a dose of 30 μM for 24 h before being exposed to three different doses of γ-IR (2, 4, and 6 Gy) [Figure 1]b. We found that CAN inhibits the proliferation and clonogenic survival of HepG2 cells at 6 Gy more than the others doses [Figure 1]b. In addition, CAN radiosensitized HepG2 cells, and their combination with 6 Gy was superior to either each treatment alone [Figure 1]c.. These data indicate that CAN potently suppresses the proliferation and clonogenic survival of HepG2 cells alone and in combination with γ-IR.
Figure 1: Canagliflozin suppresses the proliferation of HepG2 cells. (a) Dose–response curve, effective concentration of canagliflozin, and IC50. (b) Clonogenic assay (right and left panels) for canagliflozin (30 μM) in combination with different doses of gamma irradiation (2, 4, and 6 Gy), and the top line of plates refers to HepG2 cells exposed to a single shot of γ-irradiation alone (2, 4, and 6 Gy). (c) Relative cell viability (%) of HepG2 cells incubated for 24 h and exposed to 6 Gy γ-irradiation, and cellular viability was determined by MTT assay. The results are expressed as the mean and standard error of the mean of three independent experiments. Vehicle versus treatment *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey's post hoc test

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Canagliflozin competently hinders glucose uptake and glycolytic metabolism of HepG2 cancer cells in which SGLT-2 is expressed

To scrutinize the antitumor potential of CAN on human HCC cells, the present study principally assessed the expression levels of SGLT2 mRNA and protein. Cancer cells display enhanced glucose uptake and metabolism, which have been asserted for a long time. In this study, SGL-2 mRNA expression and protein levels displayed a statistically significant decrease (P < 0.001) in CAN- and CAN + γ-IR-treated HepG2 cells as compared to nontreated cells [Figure 2]a and [Figure 2]b. Contrariwise, untreated irradiated HepG2 cells (HepG2+ γ-IR) showed a marked rise (P < 0.001) in SGLT-2 mRNA expression and protein level [Figure 2]a and [Figure 2]b, paralleled by 2.3 and 1.9 folds increase in glucose uptake and lactate release, respectively, as compared to control group [Figure 2]c and [Figure 2]d. Expectedly, CAN statistically significantly diminished (P < 0.001) SGLT-2 mRNA and protein levels, as well as glucose uptake and lactate release as clearly observed in HepG2 + CAN and HepG2 + CAN + γ-IR groups, indicating that CAN curbs tumor-accelerated metabolic rate via SGLT-2 blockade [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d. Data revealed a statistically significant reduction (P < 0.05) in PDH activity in HepG2+ γ-IR group as compared to vehicle-treated HepG2 cells with a robust decrease (P < 0.001) when compared to HepG2 + CAN and HepG2 + CAN + γ-IR cells, which recorded a substantial elevation (P < 0.001) and (P < 0.01) for CAN- and CAN + γ-IR-treated HepG2 cells, respectively, as compared to untreated HepG2 cells with 2.1 and 1.6 folds change in PDH activity, respectively [Figure 2]e.
Figure 2: Canagliflozin suppresses SGLT-2 mRNA expression and protein level in irradiated HepG2 cells and consequently impairs cancer cell glycolytic metabolism. (a and b) Relative SGLT-2 mRNA expression was detected by reverse transcription-polymerase chain reaction and quantified by scanning densitometric analysis and normalized to the housekeeping gene β-actin mRNA expression, and SGLT-2 protein level was determined by ELIZA technique. (c and d) Relative glucose uptake and lactate production. HepG2 cells (2 × 105) were maintained in glucose-rich Dulbecco's minimum essential medium eagle (10-mM glucose, 1-mM glutamine, without pyruvate, containing 10% dialyzed fetal bovine serum and 100 IU/mL penicillin/streptomycin), 4 h before incubation for 24 h with the indicated doses of 30 μM and 6 Gy for canagliflozin and γ-irradiation, respectively, and then the culture medium was collected to measure the glucose and lactate concentrations. The decrease in glucose concentration or the increase in lactate concentration in medium, normalized to cell number and data, is shown in percentage (%) relative to untreated HepG2 cells. (e) Pyruvate dehydrogenase activity. The results are expressed as the mean and standard error of the mean of three independent experiments. Vehicle versus treatment *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey's post hoc test

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Canagliflozin alters intracellular adenosine triphosphate and H2O2 levels in irradiated HepG2 cells

In [Figure 3]a and [Figure 3]b, data depict that CAN and/or γ-IR efficiently impairs ATP and ROS levels in HepG2 cells. Expectedly, CAN treatment statistically significantly have reduced (P < 0.05) the intracellular ATP level in HepG2 cells as compared to untreated cells by 1.5 folds. On the same line, a considerable decrement in intracellular ATP level by 1.62 and 2.9 folds was revealed in γ-IR and CAN+ γ-IR-treated HepG2 cells, respectively, after 24 h when compared to vehicle-treated HepG2 cells [Figure 3]a. With respect to the intracellular ROS level, a surge in H2O2 level by 1.7, 2.2, and 3.1 folds was perceived in CAN-, γ-IR-, and CAN + γ-IR-treated HepG2 cells, respectively, as compared to untreated cells [Figure 3]b.
Figure 3: Effect of canagliflozin on intracellular ATP and H2O2 levels in irradiated HepG2 cells. (a) Intracellular ATP level. (b) Intracellular H2O2 level. HepG2 cells (2 × 105) were maintained in glucose-rich Dulbecco's minimum essential medium eagle (10-mM glucose, 1-mM glutamine, without pyruvate, containing 10% dialyzed fetal bovine serum and 100 IU/mL penicillin/streptomycin), 4 h before incubation for 24 h with the indicated doses of 30 μM and 6 Gy for canagliflozin and γ-irradiation, respectively, and then the cells were harvested after 24 h, washed twice, lysed, and then centrifuged to obtain cytosolic fraction in supernatant. The results are expressed as the mean and standard error of the mean of three independent experiments. Vehicle versus treatment *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey's post-hoc test

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Canagliflozin alleviates endoplasmic reticulum stress-mediated cytoprotective autophagy and promotes crosstalk between autophagy and apoptosis in irradiated HepG2 cells

To ascertain the role of the ER stress response in CAN-induced autophagy in HepG2 cells, the present study sought to investigate the interplay between autophagy and apoptosis following incubation of CAN for 24 h and exposure to γ-IR at dose of 6 Gy. Hence, mRNA expression of ER-resident transmembrane sensors of UPR was examined. The obtained results revealed a remarkable decrease in ATF-6, IRE-1α, XBP-1u, XBP-1s, and CHOP, with approximately 50% reduction in their mRNA expression in CAN-treated HepG2 cells as compared to untreated cells [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e. Despite this, γ-IR-exposed HepG2 cells showed enhanced mRNA expression of ATF-6, IRE-1α, and XBP-1s with 1.6, 1.5, and 1.5 folds, respectively, associated with statistically significant reduction (P < 0.05) in CHOP mRNA expression [Figure 4]a, [Figure 4]b, [Figure 4]c, [Figure 4]d, [Figure 4]e. Furthermore, the protein expression of JNK and beclin-1 was assessed by Western blot analysis. In γ-IR-exposed HepG2 cells, there was a substantial rise (P < 0.05) in p-JNK/t-JNK ratio, indicating the enhanced expression of active JNK protein with 1.4-fold change as compared to untreated HepG2 cells [Figure 4]f and [Figure 4]h. Contrariwise, the ratio of p-JNK/t-JNK protein expression showed a statistically significant decline (P < 0.001) in CAN- and CAN + γ-IR-treated HepG2 cells as compared to γ-IR-treated HepG2 cells. Similarly, CAN- and CAN + γ-IR-treated HepG2 cells approximately halved p-JNK/t-JNK expression ratio as compared to vehicle-treated cells [Figure 4]f and [Figure 4]h, suggesting the downregulation of active JNK protein. Beclin-1, a prominent autophagy marker, demonstrated a vigorous accretion in its protein expression with 3.2, 3.8, and 5.2 folds' change in CAN-, γ-IR-, and CAN + γ-IR-treated HepG2 cells, respectively, as compared to untreated cells [Figure 4]g and [Figure 4]h. In CAN + γ-IR-treated HepG2 cells, CAN revoked UPR-mediated ER stress and inactivated ER luminal sensors (ATF-6 and IRE-1α) as well as inhibited beclin-1-mediated cytoprotective autophagy and switched it to cytotoxic autophagy via JNK inactivation.
Figure 4: Canagliflozin modulates ER stress-mediated cytoprotective autophagy and reinforce interplay between autophagy and apoptosis in irradiated HepG2 cells. HepG2 cells (2 × 105) were maintained in glucose-rich Dulbecco's minimum essential medium eagle (10-mM glucose, 1-mM glutamine, without pyruvate, containing 10% dialyzed fetal bovine serum and 100 IU/mL penicillin/streptomycin) and incubated for 24 h with indicated doses of 30 μM and 6 Gy for canagliflozin and γ-irradiation, respectively, and then RNA was extracted from harvested cells for reverse transcription-polymerase chain reaction or 30 μg of total protein was loaded in each well for Western immunoblotting. (a-e) The relative gene expression of ATF-6, IRE-1α, XBP-1u, XBP-1s, CHOP, and β-actin was detected by quantitative reverse transcription-polymerase chain reaction and quantified by scanning densitometric analysis and normalized to housekeeping gene β-actin mRNA expression. (f and g) Fold change in p-JNK/t-JNK ratio and beclin-1 protein expression normalized to β-actin. (h) Representative Western blot analysis, sodium dodecyl sulfate-polyacrylamide of t-JNK, p-JNKThr183/Tyr185, beclin-1, and β-actin. The results are expressed as the mean and standard error of the mean of three independent experiments. Vehicle versus treatment *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey's post hoc test

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Canagliflozin disrupts the crosstalk between PI3K/AKT/GSK-3β/mTOR and Wnt/β-catenin signaling pathways in irradiated HepG2 cells

The PI3K/Akt/GSK-3β/mTOR and Wnt/β-catenin signaling pathways participates in the regulation of autophagy and apoptosis. Then, the next sought was to investigate the potential influence of CAN and/or γ-IR on those signaling pathways, which are implicated in the proliferation and invasion of HCC cells. To do so, the protein expression of PI3K/AKT/GSK-3β/mTOR and Wnt/β-catenin signaling pathways was determined. [Figure 5] reveals that γ-IR-exposed HepG2 cells showed enhanced expression of the p-PI3KTyr508 (active form), p-AktThr308 (active form), p-mTORC1ser2481 (active form), and p-PTENser380 (inactive form) with 2.2, 2.9, 2.1, and 1.3 folds' change, respectively, paralleled with a marked increase in p-GSK-3-βser9 (inactive form) and significant diminution in p-β-cateninThr41/Ser45 (inactive form), denoting positive regulation of the interaction between PI3K/AKT/GSK-3β/mTOR and Wnt/β-catenin signaling pathways following γ-IR exposure. Meanwhile, CAN and CAN + γ-IR treatment for 24 h in HepG2 cells resulted in considerable reduction in the p-PI3KTyr508, p-AktThr308, p-mTORC1ser2481, and p-GSK-3-βser9 accompanied by restoration of t-PTEN efficiency as revealed by decreased p-PTEN/t-PTEN expression ratio and t-β-catenin protein expression destabilization and cytosolic entrapment by CAN treatment [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d, [Figure 5]e, [Figure 5]f and [Figure 5]h. The increase in IGF-1 protein level in γ-IR-treated HepG2 cells [Figure 5]g, clarifying the role of paracrine IGF-1/IGF-1R-mediated dual activation of PI3K/AKT/GSK-3β/mTORC1 and Wnt/β-catenin signaling pathways by γ-IR at early time point (24 h) post γ-IR treatment of HepG2 cells. Surprisingly, CAN- and CAN + γ-IR-treated HepG2 cells produced a statistically significant decrease (P < 0.001) in IGF-1 protein level as compared to γ-IR-treated HepG2 cells and with a similar pattern (P < 0.05) as compared to untreated HCC cells [Figure 5]g, suggesting that CAN treatment for 24 h circumvented paracrine IGF-1-mediated activation of PI3K/AKT/GSK-3β/mTOR signaling pathway.
Figure 5: Canagliflozin regulates the crosstalk between PI3K/AKT/GSK-3β/mTORC1 and Wnt/β-catenin signaling pathways and prevents paracrine IGF-1-mediated activation of PI3K/AKT/mTORC1 signaling in irradiated HepG2 cells. Protein expression normalized to β-actin (a) Fold change in p-PI3K/t-PI3K ratio. (b) Fold change in p-Akt/t-Akt ratio. (c) Fold change in p-mTORC1/t-mTORC1 ratio. (d) Fold change in p-PTEN/t-PTEN ratio. (e) Fold change in p-GSK-3-β/t-GSK-3-β ratio. (f) Fold change in p-β-catenin/t-β-catenin ratio. (g) Fold change in IGF-1 protein level. (h) Representative Western blot analysis, sodium dodecyl sulfate-polyacrylamide of p-PI3KTyr508, t-PI3K, p- AktThr308, t-Akt, p-mTORC1ser2481, t- mTORC1, p-PTENser380, t-PTEN, p-GSK-3-βser9, t-GSK-3-β, p-β-cateninThr41/ser45, t-β-catenin, and β-actin. The results are expressed as the mean and standard error of the mean of three independent experiments. Vehicle versus treatment *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey's Post hoc test

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Canagliflozin increases intracellular Ca2+-mediated apoptosis via caspase-12/caspase-3 activation and downregulates p53 and BCL-2 expression in irradiated HepG2 cells

In the present study, data indicated a significant increase in intracellular Ca2+ associated with a remarkable rise in caspase-12 and caspase-3 activities in CAN-, γ-IR-, and CAN + γ-IR-treated HepG2 cells as compared to vehicle-treated cells [Figure 6]a, [Figure 6]b, [Figure 6]c. Notably, CAN-, γ-IR-, and CAN + γ-IR-treated HepG2 cells exhibited significant downregulation in BCL-2 mRNA expression as compared to untreated cells [Figure 6]e. Strikingly, the mRNA expression of p53, a well-known tumor suppressor gene, drastically reduced (P < 0.001) in CAN- and CAN + γ-IR-treated HepG2 cells comparable to γ-IR-treated HepG2 cells, which recorded statistically significant rise (P < 0.05) when compared to untreated HepG2 cells, suggesting putative upregulation of p53 induced by γ-IR [Figure 6]d. The present data indicated that CAN induced release of Ca2+ from ER lumen followed by activation of ER-resident, caspase-12, and consequently activate caspase-3 and eventually induce apoptosis.
Figure 6: Influence of canagliflozin on intracellular Ca2+ level, caspase-12, and caspase-3 activities as well as mRNA expression of p53 and BCL-2 genes in irradiated HepG2 cells. HepG2 cells were incubated for 24 h with 30 μM canagliflozin and exposed to γ-irradiation at dose of 6 Gy. (a) Fold change in intracellular Ca2+ ([Ca2+]i), data are expressed as ratiometric fluorescence of 340/380 nm (F340/F380) after incubation with Fura-2 AM (5 μM) at 37°C for 1 h, and data are shown as fold change relative to untreated HepG2 cells. (b and c) Caspase-12 and caspase-3 activities were assessed by enzyme-linked immunosorbent assay, and data are expressed as fold change relative to vehicle-treated HepG2 cells. (d and e) The relative gene expression of p53, BCL-2, and β-actin was detected by quantitative reverse transcription-polymerase chain reaction and quantified by scanning densitometric analysis and normalized to housekeeping gene β-actin mRNA expression. The results are expressed as the mean and standard error of the mean of three independent experiments. Vehicle versus treatment *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA followed by Tukey's post hoc test

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 > Discussion Top

Based on in vitro assessments, in the present study, CAN alone or combined with γ-IR inhibited the proliferation and clonogenic survival of HCC cells within a dose of CAN 30 μM and at 6Gy, respectively. Glucose metabolism reprogramming is well renowned for HCC and other cancers, but it is still an enduring challenge to realize how far this metabolism influences cancer cells.[3] Distinguished from normal cell metabolism, the cancer cell was found switching its metabolism from oxidative phosphorylation (OXPHOS) to glycolysis.[17],[18] This phenomenon is called “Warburg effect,” in which cancer cell metabolism shifts to aerobic glycolysis concomitantly with high glucose uptake and preponderance of lactate production[19],[20] in both oxygen-rich and -poor conditions with abrogation of PDH activity.[17],[21] The GLUT proteins empower glucose uptake across the plasma membrane in a rate-limiting step for glucose metabolism.[22] GLUTs have been extensively studied in a wide array of tumors.[23] Recently, SGLT-2 mRNA showed overexpression in various HCC-derived cell lines including HepG2.[17] In the present study, CAN has been shown as an anticancer agent, whereas it displayed inhibition of glucose uptake, lactate release, and restored PDH activity consistently with pronounced reduction in SGLT-2 mRNA expression and protein level. In parallel with this, Kaji et al.[17] reported that SGLT-2, a regulator of glucose reabsorption in renal tubules, is overexpressed in various HCC cell lines, and its blockade by CAN efficiently aborted cellular proliferation and survival in a dose-dependent manner. On the other hand, data of γ-IR-treated HepG2-cells revealed fortuitous upregulation in SGLT-2 mRNA and protein levels, relative glucose uptake, and lactate release concurrently with diminished PDH activity. These findings indicate that γ-IR might contribute to the glycolytic phenotype switchover in HCC cells and consequently the development of resistant cellular counterpart. In support of this notion, γ-IR was found to stimulate epidermal growth factor receptor (EGFR).[24] Additionally, SGLT is in complex and under the direct control of EGFR.[25] Otherwise, γ-IR promoted glucose metabolism via boosting glucose uptake and lactate release in hypoxic MCF-7 breast cancer cells.[16] In addition, Matsuo et al.[26] elucidated that single-dose γ-IR (5 Gy) was found to induce conversion of pyruvate to lactate in SCCVII and HT29 solid tumors, denoting that γ-IR imposed alterations in tumor metabolism. Noteworthily, PDH activity represents a turning point in tumor metabolism because its inhibition during hypoxic conditions is critical for the aberrant preferential stimulation of glycolysis in cancer cells under normoxic conditions.[21] Therefore, maintaining PDH activity during different stages of tumor development prevents the sustained transformation to the glycolytic phenotype even under normoxic conditions. Highlighting the role of SGLT-2Is on PDH activity recovery, empagliflozin improved glucose oxidation in the myocardium of nondiabetic rats with myocardial infarction through restoration of PDH activity.[27] Hence, γ-IR-induced metabolic alterations and Warburg phenotype of tumor metabolism necessitate pretreatment of cancer cells with CAN.

There is ample evidence, based on the severity of stress, referring to the switch of cell fate from autophagy to apoptosis.[28],[29] Therefore, synchronous regulation of autophagy and apoptosis is considered a successful approach for anticancer agents' development and can boost cancer therapy outcomes.[30] There is a mutual crosstalk between ER stress and autophagy.[31] In the present study, CAN blunted the ER-mediated UPR response as revealed by the depressed expression of ER stress response ATF-6 and IRE-1α pathways and their downstream effectors XBP-1s as well as the pro-apoptotic mediator of ER stress response, CHOP, suggesting the suppression of major ER stress pathways by CAN. The relationship between IRE1-α and ATF6-dependent pathways was clarified by the study of Yoshida et al.,[32] who identified that the transcription factor XBP1, a target of ATF6, as a mammalian substrate of such an unconventional mRNA splicing system, showed that only the spliced form of XBP1 can activate the UPR efficiently. Interestingly, it was previously reported that phosphorylated p38 (mitogen-activated protein kinases [MAPK]) can regulate ATF6 function via phosphorylation.[33] The study of Werle et al.[34] demonstrated that monocyte chemoattractant protein-1 (MCP-1) activates the three MAPKs: extracellular signal-regulated kinase (ERK), JNK, and p38 MAPK. MCP-1 production is increased in cancers via the constitutive activation of NF-κB.[35] Hence, we could explain the decrease in the gene expression of transmembrane ER stress sensors in our results according to the study of Nasiri-Ansari et al.,[36] who reported that CAN attenuated the progression of atherosclerosis-associated inflammatory response by lowering the expression of MCP-1. In the same context, dapagliflozin, a SGLT-2 inhibitor, was found to inhibit ER stress in HK2 renal tubular cells under high glucose influx.[37] Conversely, γ-IR-treated HepG2 cells showed enhanced expression of ATF-6 and IRE-1α and stimulated conversion of XBP-1u to the active spliced XBP-1s form, but failed to elicit ER stress-mediated apoptosis as revealed by the reduced expression of CHOP, suggesting γ-IR-induced ER stress-mediated UPR response-associated cell survival. Consistent with the present findings, Dadey et al.[38] elucidated that γ-IR at a dose of 6 Gy induced the activation of ER stress signaling via PERK and ATF-6, which contribute to adaptive survival mechanisms in glioblastoma cells.

With regard to ER stress-mediated autophagy, the present data demonstrated increase of active JNK (p-JNKThr183/Tyr185) and beclin-1 protein expression with upregulation in p53 mRNA expression as perceived in γ-IR-treated HepG2-cells. On the other side, CAN- and CAN + γ-IR-treated HepG2 cells showed reduced expression of active JNK protein paralleled by enhanced beclin-1 protein expression and reduced mRNA expression of p53. During stressful stimuli, JNK activation takes place and phosphorylates BCL-2, which showed marked downregulation in its mRNA expression induced either by CAN and γ-IR or their combination in the present study, subsequently leading to BCL-2 degradation and release of beclin-1, allowing the initiation of autophagy to retain cellular homeostasis.[39] Moreover, Cheng et al.[40] referred to the significant role of IRE-1α/JNK/beclin-1 pathway in ER stress-mediated autophagy. Furthermore, Chakradeo et al.[41] reported that γ-IR induced autophagy in Hs578t breast tumor cells, HN6 head-and-neck tumor cells, and H358 non-small cell lung cancer cells, which are p53 mutant cell lines, and attributed that γ-IR induced autophagy to p53 functionality. Therefore, γ-IR induced putative upregulation in p53 expression, which drives ER stress induced by γ-IR toward cytoprotective autophagy via IRE-1α/JNK/beclin-1 pathway, especially in repressed CHOP expression cells as revealed in the present study. In support of this mechanism, Park et al.[42] demonstrated that hemoglobin subunit epsilon-1 induces radioresistance in colorectal cancer cell lines through the induction of cytoprotective autophagy via IRE-1α/JNK/beclin-1 pathway. The exhibited effect of CAN on autophagic response in HepG2 cells could be interpreted in the view of the study conducted by Huang et al.,[43] who revealed that overexpression of Beclin-1 in U87 glioblastoma induces apoptosis via binding to Bcl-2 and Bcl-xL, followed by increase in cytochrome c into the cytosol and thereafter caspases-3/-9 activation, and suggested that the autophagy gene Beclin-1 plays an important role in the fine tuning of autophagy and apoptosis through interactions with Bcl-2 family members. The tumor suppressor gene, p53, is a key regulator of the metabolic checkpoint that is activated by nutrient starvation, so loss of p53 function renders cancer cells more prone to limited nutrient supply, then fails to downregulate energy-demanding biosynthetic processes; this leads to more depletion of essential nutrients and often ends in increased oxidative stress and cell death.[44] In p53-deficient cancer cells, they were found to be unable to execute an AMP-activated protein kinase (AMPK)- induced metabolic remodeling exploited by AMPK activators as CAN.[45] Hence, CAN treatment stabilizes p53 and might then promotes its proteosomal degradation and then avail the loss of function of p53 to make cells more vulnerable to glucose withdrawal, as revealed in the present study.

Besides its role in protein folding and assembling, ER is a master organelle accountable for cytosolic Ca2+ signals as an intracellular calcium store.[46] There is mounting evidence that the release of Ca2+ from ER and the consequent rise in cytosolic Ca2+ can play a crucial role in cell survival and apoptosis.[47] Additionally, disruption of ER-Ca2+ homeostasis occurs as an early event during many forms of apoptosis and has been implicated in the pathophysiology of several diseases, including cancer.[48] The data of current investigation displayed a pronounced elevation in intracellular Ca2+ level in CAN-, γ-IR-, and CAN + γ-IR-treated HepG2 cells as compared to vehicle-treated cells with suppression of ER stress pathways. Expectedly, a diminution in intracellular ATP level was seen in CAN-treated HepG2 cells as well as elevated intracellular H2O2 and overactivity of caspase-12/-3, which indicate apoptosis. Cancer cells, which generate ATP primarily by virtue of oxidative glycolysis, are anticipated to culminate a drop in ATP upon glucose deprivation, and eventually undergo apoptosis,[49] due to ATP depletion or sever ROS production enjoined by disrupted mitochondrial activation.[50],[51] Indeed, the reduced intracellular ATP level could be attributed to the impaired glucose uptake and glycolytic metabolism induced by CAN treatment. Alongside, a redox signaling, for example high ROS levels, enables AMPK activation.[52] In addition, the release of Ca2+ from ER induces activation of AMPK, which, in turn, harnesses UPR-associated ER transmembrane sensors but mobilizes Ca2+ into mitochondria to enhance mitochondrial OXPHOS.[53] Mitochondrial Ca2+ uptake by impinging on Krebs cycle enzymes and electron transport chain activity generates ROS signals.[54] When Ca2+ level exceeds the physiological threshold as during acute stress, mitochondrial ROS production becomes detrimental and compromises mitochondrial bioenergetics and cell functions and further ATP depletion.[55] In the same context, γ-IR induces ROS generated from water radiolysis which are then target mitochondria and cause mitochondrial dysfunction and final apoptosis. Moreover, the ROS has extremely short lifespan and a limited diffusion distance, leading to low killing efficiency to tumor cells and unsatisfactory therapeutic outcomes.[56] Then, the combination of CAN with γ-IR imposed supraphysiological levels of ROS, which, in turn, causes mitochondrial bioenergetic collapse paralleled by exacerbated ROS levels and eventually further ATP depletion and apoptosis as revealed in the present study. In pursue of this metabolic collapse, Villani et al.[10] reported that CAN could suppress proliferation of prostate and lung carcinoma independently of glucose uptake via inhibition of mitochondrial respiration.

The convergence of signaling pathways regulating apoptosis and autophagy instigates discrete modes of programmed cell death often with contradictory outcomes. Particularly, PI3K/Akt signaling axis plays a crucial role in regulating the interaction between autophagy and apoptosis.[30],[57] Once triggered, Akt disables many pro-apoptotic proteins as well as the oncogenic hub central regulator, GSK-3-β.[58] Marchand et al.[59] demonstrated that GSK-3β inhibition induces prosurvival autophagy in human pancreatic cancer cells. Various lines of evidence have revealed that abrogation of PI3K/Akt pathway with subsequent activation of GSK-3β induces autophagy and apoptosis in many cancer cell types.[30],[60],[61] In addition, the implication of Wnt/β-catenin signaling in regulating metabolic homeostasis has been reported in previous studies.[62],[63] The present study findings clearly revealed a modulation in the phosphorylation status of PI3K/Akt and activation of GSK-3β with consequent β-catenin inactivation and restoration of PTEN proficiency in CAN- and CAN + γ-IR-treated HepG2-cells. A plausible mechanism by which CAN disrupts PI3K/Akt/GSK-3β and Wnt/β-catenin signaling axis is via inhibiting the paracrine IGF-1-mediated PI3K/Akt activation via AMPK stimulation.[64] Moreover, CAN might upregulate IGF-binding proteins, an endogenous IGF-1 suppressing factor, as reported by Koike et al.[65] In this study, γ-IR induced protein expression of PI3K/Akt/mTORC1 signaling and activated β-catenin paralleled by inactivation of PTEN and GSK-3β. These findings are in agreement with those obtained by Qiao et al.[66] and Yuan et al.[67] Furthermore, preoperative radiotherapy activated cancer-associated fibroblasts and promoted colorectal cancer progression via paracrine IGF-1/IGF-1R signaling,[68] which is in accordance with the obtained results. Also, Qiao et al.[66] reported that inhibition of PI3K/Akt pathway by curcumin induced radiosensitization and apoptosis of Burkitt's lymphoma cells. Hence, inhibition of PI3K/Akt/GSK-3β and Wnt/β-catenin signaling axis by CAN necessitates pretreatment of HCC tumors with CAN before radiotherapy.

 > Conclusion Top

Taken together, our results indicate that CAN/IR combination treatment suppresses the growth of HepG2 cells through inhibiting glucose uptake, lactate release, restoring PDH activity, increasing intracellular ROS, depleting intracellular ATP, and modulating ER stress- mediated cytoprotective autophagy and switched it to cytotoxic autophagy and apoptosis. Besides, CAN treatment abrogated PI3K/AKT/GSK-3-β/mTOR and WNT/β-Catenin signaling pathways induced by γ-IR and overcome the acquired radioresistance. Thus, we recommend that therapeutic strategies for HCC should include a SGLT-2I-like CAN prior to radiotherapy to achieve enhanced curative outcomes.

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Conflicts of interest

There are no conflicts of interest.

 > References Top

El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011;365:1118-27.  Back to cited text no. 1
Bruix J, Qin S, Merle P, Granito A, Huang YH, Bodoky G, et al. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): A randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017;389:56-66.  Back to cited text no. 2
Hung MH, Chen YL, Chen LJ, Chu PY, Hsieh FS, Tsai MH, et al. Canagliflozin inhibits growth of hepatocellular carcinoma via blocking glucose- influx- induced β- catenin activation. Cell Death Dis 2019;10:420.  Back to cited text no. 3
Graham NA, Tahmasian M, Kohli B, Komisopoulou E, Zhu M, Vivanco I, et al. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death. Mol Syst Biol 2012;8:589.  Back to cited text no. 4
Martin GS. Cell signaling and cancer. Cancer Cell 2003;4:167-74.  Back to cited text no. 5
Vadlakonda L, Pasupuleti M, Pallu R. Role of PI3K-AKT-mTOR and Wnt Signaling Pathways in Transition of G1-S Phase of Cell Cycle in Cancer Cells. Front Oncol 2013;3:85.  Back to cited text no. 6
Pate KT, Stringari C, Sprowl-Tanio S, Wang K, TeSlaa T, Hoverter NP, et al. Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer. EMBO J 2014;33:1454-73.  Back to cited text no. 7
Yadav RK, Chae SW, Kim HR, Chae HJ. Endoplasmic reticulum stress and cancer. J Cancer Prev 2014;19:75-88.  Back to cited text no. 8
Su J, Zhou L, Kong X, Yang X, Xiang X, Zhang Y, et al. Endoplasmic reticulum is at the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in the pathogenesis of diabetes mellitus. J Diabetes Res 2013;2013:193461-(6 pages).  Back to cited text no. 9
Villani LA, Smith BK, Marcinko K, Ford RJ, Broadfield LA, Green AE, et al. The diabetes medication Canagliflozin reduces cancer cell proliferation by inhibiting mitochondrial complex-I supported respiration. Mol Metab 2016;5:1048-56.  Back to cited text no. 10
Weigel MT, Dahmke L, Schem C, Bauerschlag DO, Weber K, Niehoff P, et al. In vitro effects of imatinib mesylate on radiosensitivity and chemosensitivity of breast cancer cells. BMC Cancer 2010;10:412.  Back to cited text no. 11
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.  Back to cited text no. 12
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8.  Back to cited text no. 13
Mingone CJ, Gupte SA, Quan S, Abraham NG, Wolin MS. Influence of heme and heme oxygenase-1 transfection of pulmonary microvascular endothelium on oxidant generation and cGMP. Exp Biol Med (Maywood) 2003;228:535-9.  Back to cited text no. 14
Zhao CB, Shi L, Pu HH, Zhang QY. The promoting effect of radiation on glucose metabolism in breast cancer cells under the treatment of cobalt chloride. Pathol Oncol Res 2017;23:47-53.  Back to cited text no. 15
Vega A, Chacón P, Monteseirín J, El Bekay R, Alba G, Martín-Nieto J, et al. Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation. J Cell Sci 2007;120:2328-37.  Back to cited text no. 16
Kaji K, Nishimura N, Seki K, Sato S, Saikawa S, Nakanishi K, et al. Sodium glucose cotransporter 2 inhibitor canagliflozin attenuates liver cancer cell growth and angiogenic activity by inhibiting glucose uptake. Int J Cancer 2018;142:1712-22.  Back to cited text no. 17
DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The biology of cancer: Metabolic reprogramming fuels cell growth and proliferation. Cell Metab 2008;7:11-20.  Back to cited text no. 18
Warburg O. On the origin of cancer cells. Science 1956;123:309-14.  Back to cited text no. 19
Fan T, Sun G, Sun X, Zhao L, Zhong R, Peng Y. Tumor energy metabolism and potential of 3-bromopyruvate as an inhibitor of aerobic glycolysis: Implications in tumor treatment. Cancers (Basel) 2019;11:317-40.  Back to cited text no. 20
Yonashiro R, Eguchi K, Wake M, Takeda N, Nakayama K. Pyruvate dehydrogenase PDH-E1β controls tumor progression by altering the metabolic status of cancer cells. Cancer Res 2018;78:1592-603.  Back to cited text no. 21
Barron CC, Bilan PJ, Tsakiridis T, Tsiani E. Facilitative glucose transporters: Implications for cancer detection, prognosis and treatment. Metabolism 2016;65:124-39.  Back to cited text no. 22
Adekola K, Rosen ST, Shanmugam M. Glucose transporters in cancer metabolism. Curr Opin Oncol 2012;24:650-4.  Back to cited text no. 23
Dittmann K, Mayer C, Kehlbach R, Rothmund MC, Peter Rodemann H. Radiation-induced lipid peroxidation activates src kinase and triggers nuclear EGFR transport. Radiother Oncol 2009;92:379-82.  Back to cited text no. 24
Weihua Z, Tsan R, Huang WC, Wu Q, Chiu CH, Fidler IJ, et al. Survival of cancer cells is maintained by EGFR independent of its kinase activity. Cancer Cell 2008;13:385-93.  Back to cited text no. 25
Matsuo M, Kawai T, Kishimoto S, Saito K, Munasinghe J, Devasahayam N, et al. Co-imaging of the tumor oxygenation and metabolism using electron paramagnetic resonance imaging and 13-C hyperpolarized magnetic resonance imaging before and after irradiation. Oncotarget 2018;9:25089-100.  Back to cited text no. 26
Yurista SR, Silljé HH, Oberdorf-Maass SU, Schouten EM, Pavez Giani MG, Hillebrands JL, et al. Sodium-glucose co-transporter 2 inhibition with empagliflozin improves cardiac function in non-diabetic rats with left ventricular dysfunction after myocardial infarction. Eur J Heart Fail 2019;21:862-73.  Back to cited text no. 27
Zhao GX, Pan H, Ouyang DY, He XH. The critical molecular interconnections in regulating apoptosis and autophagy. Ann Med 2015;47:305-15.  Back to cited text no. 28
Liu Y, Yang B, Zhang L, Cong X, Liu Z, Hu Y, et al. Ginkgolic acid induces interplay between apoptosis and autophagy regulated by ROS generation in colon cancer. Biochem Biophys Res Commun 2018;498:246-53.  Back to cited text no. 29
Sophia J, Kowshik J, Dwivedi A, Bhutia SK, Manavathi B, Mishra R, et al. Nimbolide, a neem limonoid inhibits cytoprotective autophagy to activate apoptosis via modulation of the PI3K/Akt/GSK-3β signalling pathway in oral cancer. Cell Death Dis 2018;9:1087.  Back to cited text no. 30
Lin Y, Jiang M, Chen W, Zhao T, Wei Y. Cancer and ER stress: Mutual crosstalk between autophagy, oxidative stress and inflammatory response. Biomed Pharmacother 2019;118:109249-(10 pages).  Back to cited text no. 31
Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 2001;107:881-91.  Back to cited text no. 32
Egawa N, Yamamoto K, Inoue H, Hikawa R, Nishi K, Mori K, et al. The endoplasmic reticulum stress sensor, ATF6α, protects against neurotoxin-induced dopaminergic neuronal death. J Biol Chem 2011;286:7947-57.  Back to cited text no. 33
Werle M, Schmal U, Hanna K, Kreuzer J. MCP-1 induces activation of MAP-kinases ERK, JNK and p38 MAPK in human endothelial cells. Cardiovasc Res 2002;56:284-92.  Back to cited text no. 34
Yoshimura T. The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments. Cytokine 2017;98:71-8.  Back to cited text no. 35
Nasiri-Ansari N, Dimitriadis GK, Agrogiannis G, Perrea D, Kostakis ID, Kaltsas G, et al. Canagliflozin attenuates the progression of atherosclerosis and inflammation process in APOE knockout mice. Cardiovasc Diabetol 2018;17:106.  Back to cited text no. 36
Shibusawa R, Yamada E, Okada S, Nakajima Y, Bastie CC, Maeshima A, et al. Dapagliflozin rescues endoplasmic reticulum stress-mediated cell death. Sci Rep 2019;9:9887.  Back to cited text no. 37
Dadey DY, Kapoor V, Khudanyan A, Urano F, Kim AH, Thotala D, et al. The ATF6 pathway of the ER stress response contributes to enhanced viability in glioblastoma. Oncotarget 2016;7:2080-92.  Back to cited text no. 38
Benbrook DM, Long A. Integration of autophagy, proteasomal degradation, unfolded protein response and apoptosis. Exp Oncol 2012;34:286-97.  Back to cited text no. 39
Cheng X, Liu H, Jiang CC, Fang L, Chen C, Zhang XD, et al. Connecting endoplasmic reticulum stress to autophagy through IRE1/JNK/beclin-1 in breast cancer cells. Int J Mol Med 2014;34:772-81.  Back to cited text no. 40
Chakradeo S, Sharma K, Alhaddad A, Bakhshwin D, Le N, Harada H, et al. Yet another function of p53-the switch that determines whether radiation-induced autophagy will be cytoprotective or nonprotective: Implications for autophagy inhibition as a therapeutic strategy. Mol Pharmacol 2015;87:803-14.  Back to cited text no. 41
Park SY, Lee SJ, Cho HJ, Kim JT, Yoon HR, Lee KH, et al. Epsilon-globin HBE1 enhances radiotherapy resistance by down-regulating BCL11A in colorectal cancer cells. Cancers (Basel) 2019;11:498-514.  Back to cited text no. 42
Huang X, Qi Q, Hua X, Li X, Zhang W, Sun H, et al. Beclin 1, an autophagy-related gene, augments apoptosis in U87 glioblastoma cells. Oncol Rep 2014;31:1761-7.  Back to cited text no. 43
Flöter J, Kaymak I, Schulze A. Regulation of metabolic activity by p53. Metabolites 2017;7:21-38.  Back to cited text no. 44
Aning OA, Cheok CF. Drugging in the absence of p53. J Mol Cell Biol 2019;11:255-64.  Back to cited text no. 45
Mekahli D, Bultynck G, Parys JB, De Smedt H, Missiaen L. Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb Perspect Biol 2011;3:a004317 (30 pages).  Back to cited text no. 46
Kaufman RJ. Stress signaling from the lumen of the endoplasmic reticulum: Coordination of gene transcriptional and translational controls. Genes Dev 1999;13:1211-33.  Back to cited text no. 47
Shin DH, Leem DG, Shin JS, Kim JI, Kim KT, Choi SY, et al. Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells. J Ginseng Res 2018;42:165-74.  Back to cited text no. 48
Moley KH, Mueckler MM. Glucose transport and apoptosis. Apoptosis 2000;5:99-105.  Back to cited text no. 49
Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, et al. Inhibition of glycolysis in cancer cells: A novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer Res 2005;65:613-21.  Back to cited text no. 50
Jelluma N, Yang X, Stokoe D, Evan GI, Dansen TB, Haas-Kogan DA. Glucose withdrawal induces oxidative stress followed by apoptosis in glioblastoma cells but not in normal human astrocytes. Mol Cancer Res 2006;4:319-30.  Back to cited text no. 51
Cardaci S, Filomeni G, Ciriolo MR. Redox implications of AMPK-mediated signal transduction beyond energetic clues. J Cell Sci 2012;125:2115-25.  Back to cited text no. 52
Chauhan AS, Liu X, Jing J, Lee H, Yadav RK, Liu J, et al. STIM2 interacts with AMPK and regulates calcium-induced AMPK activation. FASEB J 2019;33:2957-70.  Back to cited text no. 53
Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003;552:335-44.  Back to cited text no. 54
Feno S, Butera G, Vecellio Reane D, Rizzuto R, Raffaello A. Crosstalk between calcium and ROS in pathophysiological conditions. Oxid Med Cell Longev 2019;2019:9324018.  Back to cited text no. 55
Zhang C, Zhao K, Bu W, Ni D, Liu Y, Feng J, et al. Marriage of scintillator and semiconductor for synchronous radiotherapy and deep photodynamic therapy with diminished oxygen dependence. Angew Chem Int Ed Engl 2015;54:1770-4.  Back to cited text no. 56
Chang CH, Lee CY, Lu CC, Tsai FJ, Hsu YM, Tsao JW, et al. Resveratrol-induced autophagy and apoptosis in cisplatin-resistant human oral cancer CAR cells: A key role of AMPK and Akt/mTOR signaling. Int J Oncol 2017;50:873-82.  Back to cited text no. 57
Nagini S, Sophia J, Mishra R. Glycogen synthase kinases: Moonlighting proteins with theranostic potential in cancer. Semin Cancer Biol 2019;56:25-36.  Back to cited text no. 58
Marchand B, Arsenault D, Raymond-Fleury A, Boisvert FM, Boucher MJ. Glycogen synthase kinase-3 (GSK3) inhibition induces prosurvival autophagic signals in human pancreatic cancer cells. J Biol Chem 2015;290:5592-605.  Back to cited text no. 59
Ko CP, Lin CW, Chen MK, Yang SF, Chiou HL, Hsieh MJ. Pterostilbene induce autophagy on human oral cancer cells through modulation of Akt and mitogen-activated protein kinase pathway. Oral Oncol 2015;51:593-601.  Back to cited text no. 60
Shao X, Lai D, Zhang L, Xu H. Induction of autophagy and apoptosis via PI3K/AKT/TOR pathways by azadirachtin A in Spodoptera litura cells. Sci Rep 2016;6:35482.  Back to cited text no. 61
Elghazi L, Gould AP, Weiss AJ, Barker DJ, Callaghan J, Opland D, et al. Importance of β-Catenin in glucose and energy homeostasis. Sci Rep 2012;2:693.  Back to cited text no. 62
Behari J, Li H, Liu S, Stefanovic-Racic M, Alonso L, O'Donnell CP, et al. β-catenin links hepatic metabolic zonation with lipid metabolism and diet-induced obesity in mice. Am J Pathol 2014; 184:3284-98.  Back to cited text no. 63
Ning J, Clemmons DR. AMP-activated protein kinase inhibits IGF-I signaling and protein synthesis in vascular smooth muscle cells via stimulation of insulin receptor substrate 1 S794 and tuberous sclerosis 2 S1345 phosphorylation. Mol Endocrinol 2010;24:1218-29.  Back to cited text no. 64
Koike Y, Shirabe SI, Maeda H, Yoshimoto A, Arai K, Kumakura A, et al. Effect of canagliflozin on the overall clinical state including insulin resistance in Japanese patients with type 2 diabetes mellitus. Diabetes Res Clin Pract 2019;149:140-6.  Back to cited text no. 65
Qiao Q, Jiang Y, Li G. Inhibition of the PI3K/Akt –NF-κB pathway with curcumin enhanced radiation- induced apoptosis in human Burkitt's lymphoma. J Pharmacol Sci 2013;121:247-56.  Back to cited text no. 66
Yuan DY, Meng Z, Xu K, Li QF, Chen C, Li KY, et al. Betulinic acid increases radiosensitization of oral squamous cell carcinoma through inducing Sp1 sumoylation and PTEN expression. Oncol Rep 2017;38:2360-8.  Back to cited text no. 67
Tommelein J, De Vlieghere E, Verset L, Melsens E, Leenders J, Descamps B, et al. Radiotherapy-activated cancer-associated fibroblasts promote tumor progression through paracrine IGF1R activation. Cancer Res 2018;78:659-70.  Back to cited text no. 68


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1]


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