All reagents used were of tissue culture or analytical grade. Tissue culture medium DMEM was purchased from HiMedia (Bombay, India). All culture media were supplemented with 20 mg/ml gentamycin, 0.1 mg/ml streptomycin, 100 IU penicillin purchased from HiMedia (Bombay, India), and 10% fetal bovine serum from HiMedia (Bombay, India), henceforth, referred to as complete medium. Medium without serum was designated as an incomplete medium. Curcumin used was procured from HiMedia (Bombay, India) and was dissolved in DMSO to prepare the stock. The stock of curcumin was further diluted in a complete medium to obtain a working solution (17). Anticancer Drugs doxorubicin and methotrexate were procured from HiMedia (Bombay, India). Authenticated hepatic carcinoma HepG2cells were procured from cell repository at National Centre for Cell Science (NCCS) (Pune, India), Government of India. The cell line was maintained in vitro in the passage as well as in cryopreserved form. The maximum passage number of cells used was less than 25; and the passage number difference in any two experiments in this investigation was less than five.

The viability of cancer cells was enumerated by a standard trypan blue dye exclusion assay. The survival of cancer cells was assayed according to a method described earlier (28). Briefly, cells incubated in 96-well plate, with culture condition indicated in the results, were stained with 0.5% (w/v) crystal violet solution containing 20% (v/v) methanol. After washing with PBS, 200 µl methanol was added to each well and incubated at room temperature in rocking condition for 20 min. Absorbance of control (A_control ) and treated (A_treated ) groups were read at 590 nm on a Multiscan EX mirotitre plate reader. Relative survival was calculated using a formula as follows:

For calculation of IC₅₀, cells were treated with various concentrations of anticancer drugs (0–20 mM) in conditions indicated in the results. After 24 h of incubation, the survival of cancer cells was estimated. IC₅₀ was calculated by a linear interpolation method using the following formula:

where A = the first point on the curve that is less than 50% inhibition, B = the first point on the curve that is greater than or equal to 50% inhibition, C = the concentration of drug that gives A% inhibition, and D = the concentration of drug that gives B% inhibition.

Metabolic activity of cells was estimated using MTT assay (14). Cancer cells (1 × 10⁵/ml) were seeded in a 96-well tissue culture plate in a complete medium with indicated concentrations of curcumin and/or standard anticancer drugs or additional glucose, as described in Results. Cell survival was measured by standard MTT assay. MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] was dissolved in PBS at a concentration of 5 mg/ml of MTT solution. A 50 µl of the solution was added to each well of the 96-well culture plate containing cells in 200 µl medium and incubated at 37°C for 4 h for the formation of dark blue formazan crystals. The medium was then carefully removed without disturbing the crystals. DMSO (50 µl) was added to each well and mixed thoroughly to dissolve the formazan crystals. The plates were then read on a Multiscan EX microplate reader (Thermo Scientific) at a wavelength of 540 nm. Metabolic activities of cells are presented as relative to control.

Dead cell percentage was enumerated with the method described earlier (29). Briefly, cancer cells were tissue culture plate in complete medium and were treated as given in results. Cells were then harvested using trypsin-EDTA, and the number of live (N_Viable ) and death cells (N_Dead ) populations was enumerated by hemocytometer and microscopy techniques using the trypan blue dye exclusion method. Death cell percent was calculated using a standard formula.

The apoptotic cell population was enumerated by a method described earlier (17). Air-dried and methanol-fixed smear of cell suspension was stained with Wright-Giemsa staining solution, mounted in glycerin, and analyzed under a light microscope (Leica, India). Morphological features associated with apoptotic cells include the contracted cell bodies, condensed, uniformly circumscribed, and densely stained chromatin, and membrane-bound apoptotic bodies containing one or more nuclear fragments. The percentage of apoptotic cells was determined by counting at least three hundred cells in at least three separate microscopic fields.

The concentration of stable nitrite NO₂, the end product from NO generation, was determined in the cell-free culture supernatant by the method described earlier (30) with slight modification based on the Griess reaction. Culture supernatants were incubated with an equal volume of Griess reagent [one part of 1% (w/v) sulfanilamide in 2.5% H3PO4 plus one part of 0.1% (w/v) naphthyl-ethylene-diamine dihydrochloride; two parts being mixed within 12 h of use and kept chilled at room temperature for 10 min in a 96-well Microtiter plate]. The absorbance at 540 nm was determined by an automatic ELISA plate reader (Thermo Scientific). Nitrite content was quantified by extrapolation from a standard curve of NaNO₂ in each experiment. In all the experiments, nitrite content in the wells containing medium without cells was also measured and subtracted.

Glucose content was measured using a commercial kit from Beacon Diagnostics Pvt. Ltd, India, based on the conversion of glucose to H₂O₂ by the action of glucose oxidase and final estimation of the generated H₂O₂ by converting it into a colored red quinone product by the action of peroxidase (14). Briefly, 10 ml of fluid sample was mixed with 1 ml of working reagent containing phosphate buffer (pH 7.4), phenol, glucose-oxidase, peroxidase, and 4-Aminoantipyrine and was incubated for 10 min at 37˚C. The final reading was taken at 505 nm. Glucose content is expressed as mM. To determine the glucose consumption, the differences of values for culture supernatant and uninoculated media were determined.

Lactate concentration was measured using an enzymatic colorimetric method (Spinreact, Granada, Spain) (17). Briefly, 1 ml sample was diluted in 200 ml 50 mM PIPES (pH 7.5) containing 4-chlorophenol (4 mM), lactate oxidase (800 U/L), peroxidase (2,000 U/L), and 4-aminophenazone (0.4 mM), followed by incubation for 10 min at room temperature, and measurement of absorbance at 505 nm was recorded. Lactate concentration was expressed in mM.

Culture supernatants of HepG2 cells (1 × 10⁵/ml) treated with curcumin in a medium containing standard or additional glucose concentration were collected after 24 h of incubation. The pH level of cell-free culture supernatants was measured using a probe of pH meter.

Induction of apoptotic mode of cell death in cells was also confirmed by quantitative determination of DNA fragmentation (29). Tumor cells were lysed in 0.5 ml lysis buffer (0.2% v/v TritonX-100 in Tris-EDTA buffer, pH 7.4) and centrifuged (13,000 g at 4°C for 10 min) to separate intact and fragmented DNA in pellet and supernatant in tubes. Trichloroacetic acid (0.5 ml of 25%) was added to each tube and mixed thoroughly using a vortex. Tubes were kept overnight at 4°C for DNA precipitation. After centrifugation (13,000 g at 4°C for 10 min), pellets were mixed with 80 µl of 5% trichloroacetic acid. DNA was hydrolyzed by heating at 90°C for 15 min, mixed with 160 µl diphenylamine (DPA) reagent prepared by dissolving the 150 mg diphenylamine in a mixture of 10 ml glacial acetic acid, 150 ml concentrated H₂SO₄, and 50 ml of acetaldehyde solution. A blank was included containing 80 µl of 5% trichloroacetic acid. The reaction mixture in tubes was allowed to develop color overnight at room temperature. Absorbance was measured at 600 nm in a Multiscan EX microplate reader (Thermo Scientific). Percent DNA fragmentation was calculated by putting the values of absorbance for a tube containing fragmented (A_frag ) or intact (A_int ) DNA in the following formula:

Cell nuclear morphology was evaluated by fluorescence microscopy following DAPI staining described earlier (31) with slight modification. Briefly, cells were washed with PBS and fixed with ice-cold methanol. Fixed cells were then stained with DAPI (300 nM) for 20 min at 4°C in the dark. Stained cells were washed briefly with PBS and were observed under a fluorescence microscope (Leica, India) using a 358 nm excitation and 460 nm emission fluorescent filter. Images were captured and transformed for Sobel edge detection. Images were then analyzed for mean intensity and standard deviation of DAPI intensities of the pixels in the image of individual nuclei using ImageJ software. The coefficient of variation was obtained by dividing the standard deviation by mean as a quantitative measure of chromatin condensation (32).

Compromised membrane integrity accompanies cell death which can be detected by PI uptake by dying cells. The method described earlier was used with slight modification (33). Briefly, cells were washed with PBS and then stained with PI (10 nM) for 30 min at 4°C in the dark. Stained cells were then washed briefly with PBS and immediately observed under a fluorescence microscope (Leica, India) using a 535 nm excitation and 617 nm emission fluorescent filter. Red fluorescence (PI-positive) in cells indicated the compromised membrane integrity and death. Cells with and without fluorescence were enumerated to calculate the percent PI-positive cells.

Drug uptake was evaluated by fluorescence microscopy following the doxorubicin staining described earlier (34) with slight modification. HepG2 cells (1 × 10⁵/ml) were seeded in the 12-well culture plate in a medium containing the concentration of glucose and curcumin as indicated in the results. After incubation of 24 h at 37°C in a CO₂ incubator, culture supernatant was removed; cells were rinsed twice with sterile PBS. Doxorubicin (25 μM) was then added to each well and incubated for 15 min at RT. After washing with sterile PBS, cells were observed under a fluorescence microscope (Leica, India) using appropriate excitation and emission filters. Images were captured, and doxorubicin fluorescence intensity was quantified by ImageJ software.

Level of ROS was evaluated by fluorescence microscopy following DCFDA staining described earlier (35) with slight modification. HepG2 cells (~1 × 10⁵/ml) were plated in the 12-well culture plate in a medium containing the indicated amount of glucose for 24 h. Cells were then washed with sterile PBS twice, and 10 μM DCFDA was added for 30 min at 37°C followed by stimulation with curcumin, doxorubicin, or both for 15 min. After washing, cells were observed under a fluorescence microscope (Leica, India) using a 535 nm excitation and 635 nm emission fluorescent filter. Images were captured and the intensity of the stain was quantified by ImageJ software.

RT-PCR analysis for the expression of indicated genes, and β-actin was carried out according to a method described earlier (8). Primer sequences for various genes are shown in Table 1 . PCR was performed for 15 min to make cDNA at 50°C. The amplification was carried out for 35 cycles with an initial denaturation at 94°C for 2 min followed by annealing (annealing temperature as per respective primer design) for 30 s and elongation at 72°C for 30 s. The samples were separated on an agarose gel (1%) containing ethidium bromide (0.3 mg/ml). Bands were visualized and analyzed on a UV-transilluminator (Biorad, Australia), and the intensity of bands was analyzed by ImageJ software. Band intensities of β-actin were used as a loading control. To comply with MIQE guidelines (36), invariable expression of β-actin was ensured by comparing the band intensities among experimental groups and obtaining coefficient of correlation not less than 0.95 between band intensity and amount of template used for PCR amplification ( Supplementary Figures 1 and 2 ).

Expression level of proteins was estimated by western blot analysis (35). Protein content in cell lysates prepared with Triton-X 100 lysis buffer was determined by Bradford assay. Equal protein contents separated on SDS-PAGE were transferred on nitrocellulose membrane followed by their probing with specific primary antibodies. Membranes were then incubated with alkaline phosphatase conjugated secondary antibody. After washing to remove unbound antibodies, protein bands were detected by using BCIP/NBT solution. Membranes containing developed bands were then scanned, and band intensities were measured by ImageJ software. Intensities of band of specific proteins were compared with band intensities of β-actin (loading control).

All the experiments were conducted at least thrice in triplicate. The statistical significance of differences between test groups was analyzed by one-tailed or two-tailed Student’s t-test as appropriate. The difference was considered significant only when the p-value was less than 0.05 in compared groups.

To enumerate the effect of elevated levels of glucose on tumor cells’ survival, HepG2 (1×10⁵/ml) cells were incubated in complete medium alone having a standard concentration of glucose (11 mM) or containing an additional gradient of glucose concentration. After incubation for 24 h, cell survival was measured. Results are shown in Figure 1A . A concentration-dependent increase in the survival of HepG2 cells was observed. Statistically significant augmentation in cell survival was observed up to 25 mM concentration of glucose. The dead cell population was also enumerated using the trypan blue dye exclusion method and results are given in Figure 1B . A decline in the percent dead cell population was recorded with an increase in the glucose concentration. Metabolic activity of cells was also found to augment by additional glucose level in a concentration-dependent manner ( Figure 1C ). For augmentation of survival and metabolic activity or decline in the dead cell population, a peak statistically significant deviation was observed in media containing 25 mM glucose. Therefore, hereafter 25 mM concentration of glucose was termed as high glucose (HG) and was used in further experiments, otherwise stated. The standard concentration of glucose in media (11 mM) was used as normal glucose (NG) concentration.

In order to evaluate the effect of HG on the apoptotic parameter, HepG2 cells were incubated in a medium containing either NG or HG levels. After 24 h of incubation, cells were harvested and processed for determination of DNA fragmentation as per the method described in Materials and Methods. As indicated in Figure 1D , the DNA fragmentation level was found to be significantly lower in the cells incubated with HG concentration.

For evaluation of the effect of elevated levels of glucose on tumor cells’ susceptibility towards cytotoxic action of standard chemotherapeutic drugs, HepG2 cells (1×10⁵/ml) were incubated for 24 h in a medium having NG or HG level and containing either doxorubicin or methotrexate in increasing concentration. After incubation, estimation of cell survival and metabolic activity and enumeration of the dead cell population were carried out using standard crystal violet assay, MTT assay, and trypan blue dye exclusion assay, respectively. Results are given in Figures 2A, B . Survival of cancer cells was found to decline with an increase in the concentration of anticancer drugs either doxorubicin or methotrexate. Observed decline of cell survival was less in cells incubated in HG medium. The survival of HepG2 cells incubated with anticancer drugs in HG-containing medium was significantly higher as compared to those incubated in NG medium containing the same concentration of either doxorubicin or methotrexate. Similar effect of anticancer drugs was observed on metabolic activity of HepG2 cells incubated in NG or HG medium. The HG was found to resist the increase in dead cell population with an increasing level of anticancer drugs as indicated in Figures 2A, B . Tumor cells treated with anticancer drugs in HG-containing medium had a significantly lower level of percent dead cell population as compared to those incubated in NG medium. Relative abundance of dead cells versus live cell was found to increase in anticancer drug–treated groups ( Supplementary Figure 3 ). However, ratio of dead and live cells was significantly lower in the cells treated with anticancer drugs in HG medium. IC₅₀ of both the anticancer drugs were estimated, and results are given in Figure 2C . HG level was found to increase the IC₅₀ of both the drugs (doxorubicin and methotrexate) against HepG2 cells ( Figure 2C ).

To determine transient or persistent nature of effect, HepG2 cells were adapted in HG medium (HG-adapted) for six frequent passages (every 3 days). Control and HG-adapted cells were then incubated in medium containing increasing concentration of doxorubicin followed by estimation of cell survival by crystal violet assay. HG-adapted cells were found significantly resistant for decline in cell survival caused by doxorubicin ( Figure 3A ). IC₅₀ of doxorubicin was found to be significantly higher against HG-adapted cells as compared to control ( Figure 3B ).

HepG2 (1×10⁵/ml) cells were incubated in NG or HG medium containing the indicated concentration of curcumin followed by estimation of cell survival and metabolic activity. Results are given in Figures 4A, B . It was found that with increasing concentration of curcumin, cell survival of HepG2 cells decreased in both NG and HG medium. Although the decline of cell survival was less in cells incubated in NG medium, there were no statistically significant differences. Metabolic activity was also found to follow similar trend except significant difference between NG and HG groups at 20 µM curcumin concentration. Relative survival after different incubation period was also determined for HepG2 cells incubated in NG or HG medium alone or containing 5 or 10 µM curcumin ( Figure 4C ). Significantly rapid increase in survival of control cells (not treated with curcumin) was observed in HG medium as compared to NG medium. Presence of curcumin resisted the time-dependent increase of cell survival both in NG or HG medium. Nevertheless, no significant difference was observed in time-dependent increase in survival of cells incubated in NG or HG medium with curcumin. Among curcumin treated groups, the decline of cell survival was insignificant after 24 h of incubation 5 µM both in NG or HG medium. In both the NG and HG medium, 5 µM curcumin was sublethal as it was found to cause no significant decline in survival of HepG2 cells after 24h. Therefore, this concentration and time of incubation were used in subsequent chemosensitization experiments, otherwise stated.

To evaluate the effect of curcumin on hyperglycemia-driven chemoresistance in tumor cells, HepG2 cells were incubated in NG or HG medium alone or containing doxorubicin or methotrexate at 15 µM (approximate to their IC₅₀ in NG medium) in the absence or presence of a sublethal concentration of curcumin. Survival, IC₅₀, and metabolic activity of HepG2 cells were estimated as per the standard method given in Materials and Methods. As indicated in Figure 4D , a significant decline in the survival of HepG2 cells treated with anticancer drugs along with curcumin was observed as compared to those treated with anticancer drugs alone both in NG and HG medium, indicating cooperation between curcumin and anticancer drugs. The difference in the level of cell survivals in NG and HG medium containing anticancer drugs in presence of a sublethal concentration of curcumin was found to be insignificant. Curcumin was also found to significantly decrease the IC₅₀ of anticancer drugs (doxorubicin and methotrexate) in both the mediums either containing NG or HG levels ( Figure 4E ). Metabolic activity of HepG2 cells followed the trend similar to observation of cell survival with same treatment ( Figure 4F ).

Dead cell population in HepG2 cells treated with doxorubicin in NG or HG medium in absence or presence of curcumin was discriminated from live cells using PI uptake assay. Cells with red fluorescence were enumerated. Representative photomicrographs and enumerated dead cell population are given in Figure 5 . Doxorubicin was found to augment the dead cell population in HepG2 cells incubated in NG medium; however, there was a lower percentage of PI-positive cells in the HG group of cells treated with doxorubicin. When cells are treated with doxorubicin in the presence of curcumin, a significant increase in fluorescent cells was observed both in medium containing NG or HG level, and there was no significant difference in percent PI-positive cells.

HepG2 cells were incubated in a medium containing normal glucose level alone or in presence of either 5 or 10 µM concentration of curcumin. Cell-free supernatants were harvested at the regular time interval as indicated. The concentration of remaining glucose was estimated and was subtracted from the initial glucose concentration to determine the amount of glucose consumed. Results are given in Figure 6 . The amount of consumed glucose was found to be increasing with the time of incubation. Both 5 and 10 µM concentrations of curcumin inhibited the glucose consumption by HepG2 cells. Glucose consumption was found to be significantly lower by HepG2 cells incubated in a curcumin-containing medium for 12 h or more. The difference in glucose consumption was found more significant when cells were incubated in a medium containing 10 µM of curcumin for 24 h or more.

To evaluate the ability of curcumin on the high level of glucose-mediated alterations in extracellular pH and lactate level, HepG2 (1×10⁵/ml) cells were incubated in medium containing a normal or high concentration of glucose in the absence or presence of curcumin (5 µM), and cell-free culture supernatants were collected. The pH of culture supernatants was estimated using a pH meter, and lactate levels were estimated as per the method given in Materials and Methods. Results are given in Table 2 . The culture supernatant of cells incubated in the HG medium was found to have significantly acidic (lower) pH as compared to those incubated in the NG medium. Curcumin treatment was found to resist the lowering of pH of culture supernatant both in NG as well in HG medium. The lactate level in the culture supernatant was found to be significantly higher for the cells incubated in the HG medium. Curcumin treatment decreases the lactate level in the culture supernatant of cells incubated either in an NG or HG-containing medium. No significant difference was found in the lactate level in the culture supernatant of cells treated with curcumin either in NG or HG medium.

Nitric oxide level was also estimated in the culture supernatant of HepG2 cells treated with curcumin in a medium either containing a normal or high level of glucose. Results are given in Table 2 . Culture supernatant of cells incubated in the HG medium had a significantly lower level of nitric oxide. Curcumin was found to significantly augment the level of nitric oxide in culture supernatants of the cells incubated in NG and HG medium. There was no significant difference observed in the culture supernatant NO level of NG or HG groups treated with a sublethal concentration of curcumin.

Tumor cells treated with doxorubicin in NG or HG medium with and without sublethal concentration of curcumin were processed for detection of ROS generation using DCFDA and observed under a fluorescent microscope. Images of fluorescence were processed using ImageJ software for the determination of mean fluorescence intensity (MFI). Results are given in Figure 7 . Doxorubicin treatment significantly augmented the level of ROS in the cells incubated either in NG or HG medium. However, the ROS level was found to be significantly lower in doxorubicin-treated cells incubated in HG medium as compared to those incubated in NG medium. Treatment of cells with doxorubicin in presence of curcumin was found to cooperatively augment the ROS level in cells of both NG and HG groups.

To evaluate whether pretreatment of curcumin can have any effect on hyperglycemia-mediated chemoresistance, HepG2 cells (1×10⁵/ml) were incubated in sublethal (5 µM) curcumin containing medium for a time indicated in the results. Cells were then washed and cultured in a medium containing 25 mM glucose (HG) with indicated concentrations of doxorubicin. After 24 h of incubation, cell survival, IC₅₀, and metabolic activity were estimated as per the standard method described in Materials and Methods. It was found that doxorubicin was able to inhibit the cell survival of HepG2 cells in a concentration-dependent manner in control as well as curcumin pretreated cells ( Figure 8A ). However, in pretreated cells, survival of doxorubicin-treated HepG2 was significantly lower as compared to cells now exposed to curcumin. Differences in the survival of doxorubicin-treated cells between control and curcumin pretreated groups were found to be significantly higher in the group pretreated for longer (12 h) duration. Curcumin pretreatment was found to significantly decrease the IC₅₀ of doxorubicin against tumor cells incubated in an HG medium in a pretreatment time-dependent manner ( Figure 8B ). Doxorubicin-mediated decline in metabolic activity was also observed in control as well as curcumin-pretreated HepG2 cells ( Figure 8C ). The rate of decline in metabolic activity by doxorubicin was significantly higher in curcumin pretreated cells as compared to control.

The effect of HG and curcumin treatment on nuclear dynamics was detected by observing the degree of chromatin condensation using DAPI staining. Cells were incubated in NG or HG medium containing doxorubicin, curcumin, or their combination for 24 h. Cells were then stained with DAPI and observed under a fluorescent microscope. Representation images are given in Figure 9A . Photomicrographs were then processed for Sobel edge detection and analyzed for the coefficient of variation (CV) of DAPI fluorescence to determine chromatin condensation. Results are given in Figure 9B . Chromatin condensation was found to be augmented in the doxorubicin-treated group as compared to the control group incubated in an NG medium. Doxorubicin was also found to augment the chromatin condensation in cells incubated in HG medium; however, the level of condensation was significantly lower as compared to the NG group. The presence of curcumin during treatment both in NG or HG medium conjoins the doxorubicin in elevating the chromatin condensation in HepG2 cells.

The effect of curcumin and hyperglycemia on the accumulation of anticancer drugs was evaluated by incubating the HepG2 cells (1×10⁵/ml) in NG or HG medium alone or containing the sublethal concentration of curcumin. Cells were then washed and incubated in a medium containing doxorubicin for drug uptake assay as indicated in Materials and Methods. After washing, cells were observed under a fluorescent microscope, and images were captured. MFI of doxorubicin in cells was estimated using ImageJ software. Results are given in Figure 10 . Cells incubated in a medium having hyperglycemic conditions have a significantly lower level of doxorubicin fluorescence. The curcumin was found to significantly heighten the doxorubicin fluorescence in cells incubated in a medium with either normal or high levels of glucose. There was no statistically significant difference observed in doxorubicin fluorescence of cells incubated in HG or NG medium containing a sublethal concentration of curcumin.

The effect of the high level of glucose and curcumin on the expression profile of various metabolic enzymes was evaluated by determining the mRNA level using RT-PCR analysis. Representative images of bands are given in Figure 11A . A densitometric scan of the bands obtained after RT-PCR and gel electrophoresis was also done, and the relative intensity of bands of genes against bands of β-actin are given in Figure 11B . It was found that HG favors the augmented expression of most of the glycolytic enzymes (HKII, PFK1, GAPDH, PKM2, and LDH-A), IDH3A, and fatty acid synthase (FASN). In contrast, the expression level of SDH was found to be significantly lower in cells incubated in a medium containing a high concentration of glucose. The sublethal concentration of curcumin was found to significantly inhibit the mRNA expression of HKII, PFK1, GAPDH, PKM2, and LDH-A in the cells incubated in a medium containing either normal or high concentrations of glucose. A similar trend was observed with the expression of IDH3A and FASN. For the expression of SDH, curcumin treatment augmented the mRNA level in cells of either NG or HG groups.

The effect of the high level of glucose and curcumin on the expression level of mRNA of various transporters, receptors, and transcription factors was also evaluated. The results are given in Figures 12A, B . Hyperglycemia was found to augment the expression of GLUT-1, MCT-1, MCT-4, HCAR-1, and MDR-1. Curcumin was found to oppose this HG level-mediated alteration in mRNA expression of cell surface molecules. Among the transcription factors and signaling molecules, an elevated level of glucose in the culture medium favors the boosted expression of HIF-1α, mTOR, MYC, and STAT3. Curcumin treatment significantly decreases the mRNA level of these molecules in cells incubated both in NG or HG medium. HG medium cells were found to have a significantly lower level of p53 mRNA which was found to be elevated with treatment with curcumin.

Expression levels of GLUT1, HKII, HIF-1α, and p53 were found to alter in HG medium ( Figure 13 ). The expression level of GLUT1, HKII, and HIF-1α was found significantly higher in HepG2 cells incubated in HG medium as compared to NG medium. Curcumin treatment was able to significantly decrease the expression of GLUT1, HKII, and HIF-1α in HepG2 cells either incubated in NG or HG medium. The protein expression level of p53 was found to significantly decline in cell incubated in HG medium. Significant augmentations in protein expression level of p53 were observed in curcumin-treated cells, both in NG or HG medium group, as compared to their respective controls.