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Metformin and asarone inhibit HepG2 cell proliferation in a high glucose environment by regulating AMPK and Akt signaling pathway

Abstract

Background

Metabolic dysregulation is one of the hallmarks of tumor cell proliferation. Evidence indicates the potential role of the 5′adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B/Akt signaling pathway in regulating cell proliferation, survival, and apoptosis. The present study explores the effect of metformin HCl and the combination of α- and β-asarone on the proliferation of HepG2 cells in the presence of high glucose levels simulating the diabetic-hepatocellular carcinoma (HCC) condition.

Results

The metformin and asarone reduced HepG2 cell viability in a dose-dependent manner and induced morphological changes as indicated by methyl thiazolyl tetrazolium (MTT) assay. The metformin and asarone arrested the cells at the G0/G1 phase, upregulated the expression of AMPK, and downregulated Akt expression in high glucose conditions as identified by the flow cytometry technique. Further, the upregulated AMPK led to a decrease in the expression of phosphoenolpyruvate carboxykinase-2 (PCK-2) and sterol regulatory element-binding protein-1 (SREBP-1).

Conclusion

The anti-proliferative effect of metformin and asarone in the diabetic-HCC condition is mediated via AMPK and Akt pathway.

Background

Epidemiologic evidence suggests diabetes mellitus (DM) as one of the potential risk factors in the progression of hepatocellular carcinoma (HCC) [1,2,3]. The complex pathophysiological relationship between HCC and DM could be due to hyperglycemia, hyperinsulinemia, insulin resistance, insulin-like growth factor (IGF)-1, obesity, or chronic inflammation with overlapping cell signaling pathways. The various signal transduction pathways are implied in uncontrolled proliferation, mutations, invasion, migration, and survival of the tumor cells [4, 5].

There are a growing number of studies that propose the 5′adenosine monophosphate-activated protein kinase (AMPK), a highly conserved heterotrimeric serine/threonine-protein kinase, is a crucial mediator as an energy sensor in all the eukaryotic cells [6]. It plays a vital role in maintaining cellular energy homeostasis in both diabetes and cancer growth [7, 8]. Activation of the AMPK signaling pathway can influence changes in the effector’s proteins involved in the regulatory process of energy metabolism, contributing to the pathogenesis of cancer [9,10,11]. Studies indicating AMPK activation correlates strongly with the reduction in cell proliferation in tumor cells as well as non-malignant cells. These effects are reported to be mediated through different mechanisms, including the modulation of cell cycle profile, autophagy, apoptosis, de novo fatty acid synthesis, and inhibition of protein synthesis [12, 13].

In addition to the AMPK signaling pathway, the Akt, a serine/threonine-protein kinase, also serves as an important pathway in multiple cellular processes that link with the increased proliferation, survival, and apoptosis of tumor cells [14, 15]. The Akt also serves as a negative regulator of AMPK, and studies have shown that the increased expression of Akt can stimulate proliferation through multiple downstream regulations affecting the cell cycle [16].

There is a body of evidence suggesting that metformin reduces tumor growth directly through the inhibition of energy metabolism and by regulation of both AMPK-dependent and independent signaling pathways [17,18,19,20]. On the other hand, alpha (α)- and beta (β)-asarone, the two major active constituents in volatile oils of Acorus calamus, showed anti-cancer effects in various cancer cell lines [21, 22]. Our previous study showed that asarone treatment attenuated the progression of HCC in diabetic conditions in a similar manner to that achieved by metformin [23]. The present study was aimed to understand whether the anti-proliferative effect of asarone is mediated via AMPK and Akt signaling pathway similar to the metformin, which was used as a reference drug. Further, we have also tried to address the possibility of the involvement of phosphoenolpyruvate carboxykinase-2 (PCK-2) and sterol regulatory element-binding protein-1 (SREBP-1) with cancer cell growth.

Methods

Drugs and reagents

Alpha-asarone (Lot # S18779; Purity 98% w/w; PubChem CID: 636822) and beta-asarone (Lot # STBF-1179 V; Purity 70% w/w; PubChem CID: 5281758) was purchased from Sigma-Aldrich Chemical Company, USA. Metformin HCl (Lot No: METI-1710010; PubChem CID: 14219) was a gift sample from Angels Pharma India Pvt. Ltd. Hyderabad, India. Polyclonal fluorescein isothiocyanate (FITC) goat anti-rabbit secondary antibody IgG was obtained from BD Biosciences, USA (Catalog No. 554020; Lot # 8199842). The primary rabbit polyclonal IgG antibodies viz., 5′adenosine monophosphate-activated protein kinase (AMPKα1; Catalog no. orb10076; Lot # A1227), protein kinase B (Akt; Catalog No. orb159889; Lot # K8668), phosphoenolpyruvate carboxykinase-2 (PCK-2; Catalog no. orb5876; Lot # A4509), and sterol regulatory element-binding protein-1 (SREBP-1; Catalog no. orb11415; Lot # A1505) were purchased from Biorbyt Ltd., Cambridge, UK. Dulbecco’s modified Eagle’s medium (DMEM low and high glucose), fetal bovine serum (FBS) heat-inactivated, trypsin-ethylenediaminetetraacetic acid (EDTA) digestion solution 1×, calcium- and magnesium-free phosphate-buffered saline (CMF-PBS), paraformaldehyde solution, methyl thiazolyl tetrazolium (MTT), and Triton X-100 were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. All other reagents used in this study were of cell culture grade obtained from commercial suppliers.

Cell culture conditions

The HCC cell line HepG2 (ATCC® HB-8065™) was obtained and grown as recommended by the American Type Culture Collection (ATCC), USA. HepG2 cells were cultured in DMEM either in normoglycemic (5.5 mM) or in hyperglycemic (25 mM) glucose conditions depending upon the experiments that contained 10% FBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich Chemical Company, USA) maintained at 37 °C in an incubator (Labwit Scientific Pty Ltd., Australia).

Cytotoxicity by MTT assay

The cytotoxic effect of metformin HCl, α-asarone, and β-asarone on the HepG2 cell line was measured separately by the MTT assay [24]. Briefly, the cells were seeded in a 96-well flat-bottom plate (Nunc™ MicroWell™, Thermo Fisher Scientific Inc., USA) with 200 μl of cell suspension containing approximately 10,000 cells/well and allowed to adhere by incubating for 24 h at 37 °C. After 24 h, the medium was discarded and replaced with a fresh medium (200 μl) containing different concentrations of metformin HCl (1.6, 3.2, 6.4, 12.8, and 25.6 mM) and α- and β-asarone (0.12, 0.24, 0.48, 0.96, and 1.92 mM) followed by incubation for another 48 h. Untreated cells were used as control. All the media was discarded, and 200 μl of methyl thiazolyl tetrazolium (MTT) solution (0.5 mg/ml in phosphate-buffered saline) was added to each well and incubated for 3 h. Further, the MTT culture medium was removed without disturbing the crystals formed and 100 μl of dimethyl sulfoxide (DMSO) was added to each well to solubilize formed formazan crystals. Absorbance was recorded at 570 nm using a microplate reader (Biobase® EL-10A, China) to calculate the percent cell viability using the following formula:

$$ \mathrm{Percent}\ \mathrm{cell}\ \mathrm{viability}=\frac{\left(\mathrm{At}-\mathrm{Ab}\right)}{\left(\mathrm{Ac}-\mathrm{Ab}\right)}\times 100 $$

where At is the absorbance value of the test compounds (metformin and asarone), Ab is the absorbance value of the blank, and Ac is the absorbance value of the control (untreated cells).

Further, the percent of the cell viability against the concentration of each test compound was plotted and the inhibitory concentration 50 (IC50) value was generated from the dose-response curve.

Experimental study design

The IC50 values of metformin HCl and α- and β-asarone were 12.87, 0.61, and 0.73 mM, respectively, and this represents one half of the IC50 value generated from the MTT assay (Table 1). Further, the HepG2 cells were divided into four groups: the normoglycemic (NG) cultured in normal glucose of 5.5 mM, the hyperglycemic (HG) cultured in high glucose of 25 mM, the HG group treated with metformin HCl (12.87 mM), and the HG group treated with a combined dose of asarone (0.61 mM (α)-asarone; 0.73 mM (β)-asarone).

Table 1 The IC50 values of metformin HCl and α- and β-asarone based on the MTT assay and dose selected for further studies

Cell cycle analysis

The phases of the cell cycle were analyzed by measuring the amount of propidium iodide (PI)-labeled DNA staining in ethanol-fixed cells, as described elsewhere [25]. In brief, HepG2 cells (5 × 105 cells/well) were seeded in a 6-well plate (Nunc™, Thermo Fisher Scientific Inc., USA) and allowed to adhere for 24 h at 37 °C. Thereafter, fresh DMEM medium containing 10% FBS was cultured with NG, HG, HG + Metformin, and HG + Asarone for 16 h. Subsequently, the trypsinized cells were fixed (70% chilled ethanol at 4 °C for 30 min), stained with PI-RNase solution (BD Biosciences, USA), and incubated at room temperature in the dark for 30 min. Further, the stained cells were collected and mixed well for analysis. A cytomics FC500 flow cytometer (Beckman-Coulter, USA) equipped with CXP software was used to collect the data. The analysis of results was made by measuring the forward scatter (FS) and side scatter (SS) for the identification of the single cells. Further, pulse shape processing was enabled to exclude all the clumps and doublet cells for the analysis by using pulse area vs. pulse width depending upon the experiment. Finally, the data were evaluated by using an algorithm (fit Gaussian curves) available in the FlowJo v10.0.7 software, and the PI-stained histogram plots represent the percentage of cells in different phases of the cell cycle of an experiment.

Expression of AMPKα1, Akt, PCK-2, and SREBP-1

The quantitative analysis of the expression of the intracellular markers was performed by flow cytometry using a 6-well plate. The cells were cultured at a density of 3 × 105 cells/2 ml and allowed to adhere for 24 h at 37 °C. The next day, the medium was discarded and washed with 1000 μl of 1× phosphate-buffered saline (PBS) and the cells were treated with metformin and asarone; if not, they are served as a control. The drug-treated media were removed from all the wells, transferred into a 5-ml centrifuge tube, and washed with 500 μl PBS. The PBS was then removed and transferred to the same centrifuge tube. Further, 200 μl of the trypsin-EDTA solution was added to all the wells and incubated for 4 min at 37 °C. Finally, the cells were harvested directly into the centrifuge tube after transferring back the medium from the respective wells. Thereafter, the cells containing the media were centrifuged at 300×g at 25 °C for 5 min. The supernatant was decanted carefully, treated with 500 μl of 2% paraformaldehyde (PFA) solution, and incubated for 20 min at room temperature. Cells were further mixed with 1000 μl of 1× PBS to dilute PFA solution and centrifuged at 300×g at 25 °C for 5 min. After decanting the supernatant, cells were permeabilized by treatment with 500 μl of 0.1% Triton X-100, mixed well, and incubated for another 10 min. Afterwards, cells were mixed with 1000 μl of 0.5% BSA solution for settlement and centrifuged at 200×g at 25 °C for 5 min and the supernatant was decanted. Further, 0.5% BSA solution in 1× PBS was added with 6 μl of primary antibody (AMPKα1, Akt, PCK-2, and SREBP-1) not exceeding the final volume more than 100 μl, mixed well, and incubated at 25 °C for 1 h. After incubation, it was washed with 0.5% BSA solution, resuspended with 500 μl of 0.5% BSA in 1× PBS, and added with 6 μl of the secondary antibody and incubated. After 1 h of incubation in a dark room at 25 °C, cells were centrifuged and decanted. Finally, cells were resuspended in 500 μl of 0.5% BSA, mixed well, and analyzed. A cytomics FC500 flow cytometer (Beckman-Coulter, USA) equipped with CXP software was used to collect the data. The analysis of the results was carried out, as explained previously. The higher the value of geometric mean fluorescence intensity (GMFI), the higher the expression of individual markers of the cells as a population.

Statistical analysis

Statistical analysis was performed using statistical software GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Data from the individual experiments are represented as mean ± SEM and were conducted in triplicate. The results obtained were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test, and p ˂ 0.05 were considered as significant.

Results

Metformin and asarone inhibit the growth and modify the morphology of HepG2 cells

Metformin HCl (1.6 to 25.6 mM), α-asarone (0.12 to 1.92 mM), and β-asarone (0.48 to 1.92 mM) inhibited the cell viability of HepG2 cells in a concentration-dependent manner compared to untreated cells (Fig. 1A-C). The original morphology of the cells in the untreated group had a high density with adherent cell colonies, wherein few cells piled up, with a multilayered appearance. Following treatment with metformin HCl, α-asarone, and β-asarone, the cell morphology was characterized by low density, small round bodies due to shrinkage and the detachment of non-viable cells in a dose-dependent manner (Fig. 1D). The results are representative of experiments performed on three different occasions.

Fig. 1
figure 1

Metformin and asarone inhibit the growth and modify the morphology of HepG2 cells. AC Cells were treated with metformin HCl, α-asarone, and β-asarone as indicated for 48 h and the cell viability was determined by MTT assay. D Representative images to show the morphology of HepG2 cells under an inverted microscope. The scale of the bar is 100 μm. a Untreated group; the morphology of HepG2 cells had a high density with adherent cell colonies, whereas, in the treatment group, metformin HCl (b) and asarone (c, d) at higher concentration showed lower density with a small round body. Values represent mean ± SEM of an experiment done in triplicate; one-way ANOVA followed by Bonferroni test, where *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the untreated (UT) group

The combined effect of α- and β-asarone inhibits the cell proliferation

The corresponding MTT results showed that the cell proliferation of α- or β-asarone was inhibited compared to the untreated cells (p < 0.01) and that the combined effect of α- and β-asarone (0.61 mM + 0.73 mM) was better (p < 0.05) than the single effect of α- or β-asarone (0.61 mM or 0.73 mM) (Fig. 2).

Fig. 2
figure 2

The combined cell viability activity of α + β-asarone (0.61 mM + 0.73 mM) was more effective than the single entity of either α- or β-asarone (0.61 mM or 0.73 mM) when compared to the untreated (UT) group. Values represent mean ± SEM of an experiment done in triplicate; one-way ANOVA followed by Bonferroni test, where *p < 0.05, **p < 0.01, and ***p < 0.001 when compared as indicated

Metformin and asarone block HepG2 cells in the G0/G1 phase in hyperglycemic condition

We observed that in HepG2 cells, the percentage of the cells in the sub G1 phase was less in high glucose than in normal glucose. Metformin and asarone treatment arrested the cells in the G0/G1 phase and reduced the cell count in S-phase cultured in high glucose concentration. Further, metformin retained the cells in the sub G1 population stage (Fig. 3a, b). These results indicate that metformin and asarone inhibit cell proliferation at the G0/G1 phase of the cell cycle.

Fig. 3
figure 3

Metformin and asarone block HepG2 cells in G0/G1 phase in hyperglycemic condition a Cell cycle profile of HepG2 cells cultured in normal glucose (NG; 5.5 mM), high glucose (HG; 25 mM), HG + Metformin HCl, and HG + Asarone for 16 h and stained with PI-RNase was determined by flow cytometry. b Bar diagrams represent the percentage (%) of cells in different phases of the cell cycle

Metformin and asarone inhibit the expression of PCK-2 and SREBP-1 via the AMPKα1 pathway

Based on the observation that metformin and asarone treatment induces the cytotoxicity of HepG2 cells in high glucose conditions, we measured whether this is a consequence of altered cellular energy homeostasis. The PCK-2 and SREBP-1 are the downstream regulators in the AMPK pathway, which regulate gluconeogenesis, glucose uptake, and de novo lipogenesis. Flow cytometry analysis revealed that AMPKα1 expression was enhanced by both metformin and asarone. Figures 4, 5, and 6 indicate the flow cytometry histogram events and geometric mean fluorescence intensity (GMFI) depicting the expression of respective markers in HepG2 cells. A significant increase in the expression of AMPKα1 was observed indicated by the increase in the GMFI for metformin (p < 0.01) and asarone (p < 0.001) compared with the negative control (Fig. 6). Further, it was observed that the expression of PCK-2 (p < 0.001; p < 0.001) and SREBP-1 (p < 0.01; p < 0.001) decreased in the metformin- and asarone-treated groups (Figs. 4 and 5).

Fig. 4
figure 4

The treatment of high glucose-promoted HepG2 cells with metformin and asarone downregulates the expression of PCK-2. a A representative flow cytometry histogram events depicting the expression of PCK-2 in normal glucose (NG; 5.5 mM), high glucose (HG; 25 mM), HG + Metformin HCl, and HG + Asarone. b Bar diagrams represent geometric mean fluorescence intensity (GMFI) of PCK-2 in HepG2 cells. Values represent mean ± SEM of an experiment done in triplicate; one-way ANOVA followed by Bonferroni test, where ***p < 0.001 compared to the normal glucose (NG) group and ###p < 0.001 compared to the high glucose (HG) group

Fig. 5
figure 5

The treatment of high glucose-promoted HepG2 cells with metformin and asarone downregulates the expression of SREBP-1. a A representative flow cytometry histogram events depicting the expression of SREBP-1 cultured in normal glucose (NG; 5.5 mM), high glucose (HG; 25 mM), HG + Metformin HCl, and HG + Asarone. b Bar diagrams represent geometric mean fluorescence intensity (GMFI) of SREBP-1 in HepG2 cells. Values represent mean ± SEM of an experiment done in triplicate; one-way ANOVA followed by Bonferroni test, where ##p < 0.01 and ###p < 0.001 compared to the high glucose (HG) group

Fig. 6
figure 6

The treatment of high glucose-promoted HepG2 cells with metformin and asarone upregulates the expression of AMPKα1. a A representative flow cytometry histogram events depicting the expression of AMPKα1 cultured in normal glucose (NG; 5.5 mM), high glucose (HG; 25 mM), HG + Metformin HCl, and HG + Asarone. b Bar diagrams represent geometric mean fluorescence intensity (GMFI) of AMPKα1 in HepG2 cells. Values represent mean ± SEM of an experiment done in triplicate; one-way ANOVA followed by Bonferroni test, where ##p < 0.01 and ###p < 0.001 compared to the high glucose (HG) group

Metformin and asarone decrease the expression of Akt

The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), a prototypic signaling pathway, is increasingly implicated in carcinogenesis and plays a key role in the regulation of the growth and proliferation of cells. The change in the glucose concentration did not have any effect on the expression of Akt in HepG2 cells. However, a significant decrease in the expression levels of Akt was observed in metformin (p < 0.01) and asarone (p < 0.001) groups compared to untreated cells as indicated by GMFI (Fig. 7).

Fig. 7
figure 7

The treatment of high glucose-promoted HepG2 cells with metformin and asarone downregulates the expression of Akt. a A representative flow cytometry histogram events depicting the expression of Akt cultured in normal glucose (NG; 5.5 mM), high glucose (HG; 25 mM), HG + Metformin HCl, and HG + Asarone. b Bar diagrams represent geometric mean fluorescence intensity (GMFI) of Akt in HepG2 cells. Values represent mean ± SEM of an experiment done in triplicate; one-way ANOVA followed by Bonferroni test, where ##p < 0.01 and ###p < 0.001 compared to the high glucose (HG) group

Discussion

Many experimental as well as epidemiological studies suggest that the higher plasma glucose level might play a crucial role in cancer development and progression [26,27,28,29]. However, the involvement of regulatory pathways between the effect of hyperglycemia (HG) and hepatic cancer remains largely unexplored. In the present study, we attempted to explore the effect of metformin and asarone in abating the proliferation of HepG2 cells in high glucose conditions. The results of this study suggest that metformin and asarone suppress the hepatic cancer cell viability and arrested the cell cycle at the G0/G1 phase by AMPK activation and Akt inhibition.

Studies, including our own, suggest that higher blood glucose concentration may be associated with the increased risk of cancers [23, 30,31,32,33]. The increased proliferation of HepG2 cells at elevated glucose levels may be due to glucose toxicity resulting from increased production of reactive oxygen species (ROS) and alteration in the expression of genes [4, 34]. Other work reported that this could be due to a favorable environment for the cancer-signaling pathway leading to proliferation, mutations, invasion, migration, and survival [4]. The AMPK and Akt signaling pathways, along with their downstream regulators, are indicated to play an important role in the regulation of cancer as well as glucose metabolism [35].

Classically, the 5′adenosine monophosphate-activated protein kinase (AMPK) has been identified as a central metabolic sensor playing a significant role in energy homeostasis. However, the emerging evidence suggests AMPK is a possible metabolic tumor suppressor and has been implicated in preventing and treating tumor growth [36,37,38]. The AMPK activation can regulate various tissue- and cell-specific downstream markers responsible for proliferation, autophagy, and apoptosis [12, 13, 39]. The observed anti-proliferative actions of metformin and asarone in a high glucose environment and changes of AMPKα1 expression are in accordance with many other studies that correlate with AMPK activation and inhibition of tumor growth [9,10,11]. These findings suggest that the HepG2 cell line proliferation is inversely correlated to AMPK activity in high glucose conditions.

The evidence indicates the altered expression of gluconeogenic enzymes during HCC growth and differentiation, in particular, the phosphoenolpyruvate carboxykinase (PCK or PEPCK) genes. The metformin and asarone treatment decreased the expression of PCK-2 (a downstream regulator of AMPK), a gluconeogenic factor in the liver that plays a role in the regulation of the gluconeogenesis process by catalyzing the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) [40, 41]. Elevated expression of the PCK-2 gene is found in different cancers and is linked to increased anabolic metabolism, thereby playing a vital role in cancer cell proliferation [42, 43]. The observed upregulation of PCK-2 expression in our study could be due to an increase in glucose uptake and utilization, supporting the anabolic metabolism and promoting cell proliferation. In contrast, the expression of PCK-2 in the metformin and asarone treatment was downregulated, indicating the involvement of the glucose metabolism pathway in suppressing the growth of cancer cells. The earlier work suggested the role of PCK or PEPCK in increasing the glutamine and glucose concentration, thus linking anabolic pathways to cancer cell proliferation both in vivo and in vitro [44]. Furthermore, Akt also regulates the PCK or PEPCK expression mediated through phosphorylation of cyclic AMP response element-binding/Forkhead box protein O1 (CREB/FOXO1) transcriptional activity [45].

Furthermore, both AMPK and Akt are the upstream regulator of sterol regulatory element-binding protein-1 (SREBP-1) and can directly phosphorylate SREBP-1. Recently, SREBP-1, which gets upregulated in different types of cancer, has been linked to play a role in the oncogenic signaling-mediated glucose uptake and de novo lipogenesis [46,47,48]. The pharmacological targeting of SREBP-1 has revealed to significantly inhibit the glioblastoma cell growth, suggesting it as a novel molecular target in cancer [49]. In addition to regulation by AMPK, SREBP-1 has shown to be activated by the Akt/PI3K prototypic survival signaling pathway in different types of cancer. Collectively, it demonstrated the role of the Akt/PI3K signaling pathway via SREBP-1 integrating lipogenesis and glucose metabolism for rapid cancer progression [49,50,51]. In our study too, metformin and asarone decreased SREBP-1 expression in a high glucose-induced proliferation of HepG2 cells, indicating the involvement of the AMPK/SREBP-1 or Akt/SREBP-1 signaling pathway.

The deregulation of the Akt/PI3K prototypic survival signaling pathway also plays an important role in cell proliferation. Evidence indicates that abnormal activation of the Akt/PI3K signaling pathway frequently occurs in HCC patients [14,15,16]. In this study, metformin and asarone treatment significantly decreased the expression level of Akt, thereby suppressing HepG2 cell proliferation. These findings are supported by other workers who suggest that metformin inhibited the growth of various cancer cells through inhibition of Akt [52,53,54].

The observed antagonistic relationship between AMPK and Akt and their impact on the proliferation of cancer cells is in concurrence with earlier studies by other workers. The Akt/PI3K mediated carcinogenesis and tumor progression gets upregulated due to the mammalian target of rapamycin (mTOR) signaling pathway, which in turn is activated due to deregulation of the AMPK [35, 55, 56]. Besides this, the deregulation of the AMPK disturbs the insulin signaling pathway and results in elevated levels of insulin-like growth factor-1/insulin, a common pathway for both HCC and diabetic conditions [57].

Conclusion

The metformin and asarone inhibit the HepG2 cancer cell proliferation, specifically at the G0/G1 phase of the cell cycle in a high glucose environment, which is attributable to the regulation of AMPK and Akt signaling pathways (Fig. 8). Further, the inhibition of PCK-2 and SREBP-1 indicates the link between the glucose metabolic pathway and cancer cell growth. However, the potential limitations of the study include the possibility of adopting other molecular biological methods to verify our findings, along with the role of specific activators or inhibitors in high glucose-induced HepG2 cell proliferation through modulation of both the AMPK and Akt signaling pathways. Further, the investigation about the possibility of involvement of multiple pathways is essential to elucidate the molecular mechanism/s of metformin and asarone in controlling the progression of cancer.

Fig. 8
figure 8

Schematic representation of the mechanism by which metformin and asarone cause G0/G1 arrest by regulation of AMPK via PCK-2 and SREBP-1 as well as Akt signaling pathway in a high glucose environment in HepG2 cells

Availability of data and materials

All data and materials are available upon request.

Abbreviations

AMPK:

5′Adenosine monophosphate-activated protein kinase

BSA:

Bovine serum albumin

DM:

Diabetes mellitus

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Fetal bovine serum

GMFI:

Geometric mean fluorescence intensity

HCC:

Hepatocellular carcinoma

HG:

Hyperglycemic/high glucose

MTT:

Methyl thiazolyl tetrazolium

NG:

Normoglycemic/normal glucose

PCK-2:

Phosphoenolpyruvate carboxykinase-2

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde solution

PI3K:

Phosphatidylinositol 3-kinase

SREBP-1:

Sterol regulatory element-binding protein-1

References

  1. Davila JA, Morgan RO, Shaib Y, McGlynn KA, El-Serag HB (2005) Diabetes increases the risk of hepatocellular carcinoma in the United States: a population-based case control study. Gut 54:533–539

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Vecchia CA, Negri E, Decarli A, Franceschi S (1997) Diabetes mellitus and the risk of primary liver cancer. Int J Cancer 73:204–207

    PubMed  Article  Google Scholar 

  3. Yuan JM, Govindarajan S, Arakawa K, Yu MC (2004) Synergism of alcohol, diabetes, and viral hepatitis on the risk of hepatocellular carcinoma in blacks and whites in the U.S. Cancer 101:1009–1017

    PubMed  Article  Google Scholar 

  4. Jimenez CG, Garcia-Martinez JM, Chocarro-Calvo A, De la Vieja A (2014) A new link between diabetes and cancer: enhanced WNT/b-catenin signaling by high glucose. J Mol Endo 52:R51–R66

    Article  CAS  Google Scholar 

  5. Giovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA et al (2010) Diabetes and cancer: a consensus report. CA Cancer J Clin 60:207–221

    PubMed  Article  Google Scholar 

  6. Hardie DG, Carling D, Carlson M (1998) The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 67:821–855

    CAS  PubMed  Article  Google Scholar 

  7. Jones RG, Thompson CB (2009) Tumor suppressors and cell metabolism: a recipe for cancer growth. Genes Dev 23:537–548

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. Li CI, Chen HJ, Lai HC, Liu CS, Lin WY, Li TC et al (2015) Hyperglycemia and chronic liver diseases on risk of hepatocellular carcinoma in Chinese patients with type 2 diabetes-National cohort of Taiwan Diabetes Study. Int J Cancer 136:2668–2679

    CAS  PubMed  Article  Google Scholar 

  9. Li W, Saud SM, Young MR, Chen G, Hua B (2015) Targeting AMPK for cancer prevention and treatment. Oncotarget 6:7365–7378

    PubMed  PubMed Central  Article  Google Scholar 

  10. Shackelford DB, Shaw RJ (2009) The LKB1-AMPKpathway: metabolism and growth control in tumor suppression. Nat Rev Cancer 9:563–575

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Mihaylova MM, Shaw RJ (2011) The AMPK signaling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 13:1016–1023

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Motoshima H, Goldstein BJ, Igata M, Araki E (2006) AMPK and cell proliferation-AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol 574:63–71

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Liang J, Shao SH, Xu ZX, Hennessy B, Ding Z, Larrea M et al (2007) The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat Cell Bio 9:218–224

    CAS  Article  Google Scholar 

  14. Manning BD, Cantley LC (2007) AKT/PKB signaling: navigating downstream. Cell 129:1261–1274

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Vivanco I, Sawyers CL (2002) The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2:489–501

    CAS  PubMed  Article  Google Scholar 

  16. Zhou Q, Lui VWY, Yeo W (2011) Targeting the PI3K/Akt/mTOR pathway in hepatocellular carcinoma. Future Oncol 7:1149–1167

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Zhang H, Gao C, Fang L, Zhao HC, Yao SK (2013) Metformin and reduced risk of hepatocellular carcinoma in diabetic patients: a meta-analysis. Scand J Gastroenterol 48:78–87

    CAS  PubMed  Article  Google Scholar 

  18. DePeralta DK, Wei L, Ghoshal S, Schmidt B, Lauwers GY, Lanuti M et al (2016) Metformin prevents hepatocellular carcinoma development by suppressing hepatic progenitor cell activation in a rat model of cirrhosis. Cancer 122:1216–1227

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J et al (2001) Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167–1174

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Saraei P, Asadi I, Kakar MA, Moradi-Kor N (2019) The beneficial effects of metformin on cancer prevention and therapy: a comprehensive review of recent advances. Cancer Manag Res 11:3295–3313

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. Das BK, Swamy AHMV, Koti BC, Gadad PC (2019) Experimental evidence for use of Acorus calamus (asarone) for cancer chemoprevention. Heliyon 5:e01585

    PubMed  PubMed Central  Article  Google Scholar 

  22. Chellian R, Pandy V, Mohamed Z (2017) Pharmacology and toxicology of α- and β-asarone: a review of preclinical evidence. Phytomedicine 32:41–58

    CAS  PubMed  Article  Google Scholar 

  23. Das BK, Choukimath SM, Gadad PC (2019) Asarone and metformin delays experimentally induced hepatocellular carcinoma in diabetic milieu. Life Sci 230:10–18

    CAS  PubMed  Article  Google Scholar 

  24. Bahuguna A, Khan I, Bajpai V, Kang S (2017) MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh J Pharm 12:115–118

    Article  Google Scholar 

  25. Pozarowski P, Darzynkiewicz Z (2004) Analysis of cell cycle by flow cytometry. Methods Mol Biol 281:301–311

    CAS  PubMed  Google Scholar 

  26. Laguna JC, Alegret M, Roglans N (2014) Simple sugar intake and hepatocellular carcinoma: epidemiological and mechanistic insight. Nutrients 6:5933–5954

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. Stattin P, Bjor O, Ferrari P, Lukanova A, Lenner P, Lindahl B et al (2007) Prospective study of hyperglycemia and cancer risk. Diabetes Care 30:561–567

    PubMed  Article  Google Scholar 

  28. Han H, Zhang T, Jin Z, Guo H, Wei X, Liu Y et al (2017) Blood glucose concentration and risk of liver cancer: systematic review and meta-analysis of prospective studies. Oncotarget 8:50164–50173

    PubMed  PubMed Central  Article  Google Scholar 

  29. Duan W, Shen X, Lei J, Xu Q, Yu Y, Li R et al (2014) Hyperglycemia, a neglected factor during cancer progression. Biomed Res Int 2014:461917

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  30. Park EJ, Lee JH, Yu GY, He G, Ali SR, Holzer RG et al (2010) Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell 140:197–208

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Pollak M (2009) Do cancer cells care if their host is hungry? Cell Metab 9:401–403

    CAS  PubMed  Article  Google Scholar 

  32. Algire C, Zakikhani M, Blouin MJ, Shuai JH, Pollak M (2008) Metformin attenuates the stimulatory effect of a high-energy diet on in vivo LLC1 carcinoma growth. Endor Relat Cancer 15:833–839

    CAS  Article  Google Scholar 

  33. Santisteban GA, Ely JT, Hamel EE, Read DH, Kozawa SM (1985) Glycemic modulation of tumor tolerance in a mouse model of breast cancer. Biochem Biophys Res Commun 132:1174–1179

    CAS  PubMed  Article  Google Scholar 

  34. Masur K, Vetter C, Hinz A, Tomas N, Henrich H, Niggemann B et al (2011) Diabetogenic glucose and insulin concentrations modulate transcriptome and protein levels involved in tumour cell migration, adhesion and proliferation. British J Cancer 104:345–352

    CAS  Article  Google Scholar 

  35. Zhao Y, Hu X, Liu Y, Dong S, Wen Z, He W et al (2017) ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway. Mol Cancer 16:79

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  36. Hardie DG, Hawley SA, Scott JW (2006) AMP-activated protein kinase-development of the energy sensor concept. J Physiol 574:7–15

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Xiang X, Saha AK, Wen R, Ruderman NB, Luo Z (2004) AMP-activated protein kinase activators can inhibit the growth of prostate cancer cells by multiple mechanisms. Biochem Biophys Res Commun 321:161–167

    CAS  PubMed  Article  Google Scholar 

  38. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD (2005) Metformin and reduced risk of cancer in diabetic patients. BMJ 330:1304–1305

    PubMed  PubMed Central  Article  Google Scholar 

  39. Jiang X, Tan HY, Teng S, Chan YT, Wang D, Wang N (2019) The role of AMP-activated protein kinase as a potential target of treatment of hepatocellular carcinoma. Cancers 11:647

    CAS  PubMed Central  Article  PubMed  Google Scholar 

  40. Phielix E, Szendroedi J, Roden M (2011) The role of metformin and thiazolidinediones in the regulation of hepatic glucose metabolism and its clinical impact. Trends Pharmacol Sci 32:607–616

    CAS  PubMed  Article  Google Scholar 

  41. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M, Andreelli F (2012) Cellular and molecular mechanisms of metformin: an overview. Clin Sci 122:253–270

    CAS  PubMed Central  Article  PubMed  Google Scholar 

  42. Yang J, Kalhan SC, Hanson RW (2009) What is the metabolic role of phosphoenolpyruvate carboxykinase? J Biol Chem 284:27025–27029

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Vincent EE, Sergushichev A, Griss T, Gingras MC, Samborska B, Ntimbane T et al (2015) Mitochondrial phosphoenolpyruvate carboxykinase regulates metabolic adaptation and enables glucose-independent tumor growth. Mol Cell 60:195–207

    CAS  PubMed  Article  Google Scholar 

  44. Montal ED, Dewi R, Bhalla K, Ou L, Hwang BJ, Ropell AE et al (2015) PEPCK coordinates the regulation of central carbon metabolism to promote cancer cell growth. Mol Cell 60:571–583

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. He L, Li Y, Zeng N, Stiles BL (2020) Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2. Biochem J 477:1021–1031

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Guo D, Bell EH, Mischel P, Chakravarti A (2014) Targeting SREBP-1-driven lipid metabolism to treat cancer. Curr Pharm Des 20:2619–2626

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Jeon SM (2016) Regulation and function of AMPK in physiology and diseases. Exp Mol Med 48:e245

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. Nickels JT Jr (2018) New links between lipid accumulation and cancer progression. J Biol Chem 293:6635–6636

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. Guo D, Prins RM, Dang J, Kuga D, Iwanami A, Soto H et al (2009) EGFR signaling through an Akt-SREBP-1-dependent, rapamycin-resistant pathway sensitizes glioblastomas to antilipogenic therapy. Sci Signal 2:ra82

    PubMed  PubMed Central  Article  Google Scholar 

  50. Guo D, Reinitz F, Youssef M, Hong C, Nathanson D, Akhavan D et al (2011) An LXR agonist promotes glioblastoma cell death through inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent pathway. Cancer Discov 1:442–456

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. Porstmann T, Griffiths B, Chung YL, Delpuech O, Griffiths JR, Downward J et al (2005) PKB/Akt induces transcription of enzymes involved in cholesterol and fatty acid biosynthesis via activation of SREBP. Oncogene 24:6465–6481

    CAS  PubMed  Article  Google Scholar 

  52. Yung MM, Chan DW, Liu VW, Yao KM, Ngan HY (2013) Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FOXM1 signaling cascade. BMC Cancer 13:327

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. Karnevi E, Said K, Andersson R, Rosendahl A (2013) Metformin-mediated growth inhibition involves suppression of the IGF-I receptor signaling pathway in human pancreatic cancer cells. BMC Cancer 13:235

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Zhang F, Chen H, Du J, Wang B, Yang L (2018) Anticancer activity of metformin, an antidiabetic drug, against ovarian cancer cells involves inhibition of cysteine-rich 61 (cyr61)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. Med Sci Monit 24:6093–6101

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K et al (2003) A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8:65–79

    CAS  PubMed  Article  Google Scholar 

  56. Morgensztern D, McLeod HL (2005) PI3K/Akt/mTOR pathway as a target for cancer therapy. Anticancer Drugs 16:797–803

    CAS  PubMed  Article  Google Scholar 

  57. Takano A, Usui I, Haruta T, Kawahara J, Uno T, Iwata M et al (2001) Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin. Mol Cell Biol 21:5050–5062

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

The authors acknowledge CellKraft Biotech Pvt. Ltd. Bangalore, Karnataka, India, for providing the facilities to conduct the research study.

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BKD and PCG designed all the experiments and wrote the manuscript. BKD has investigated the experiments and analyzed the data. RMK has reviewed and edited the manuscript. PCG was associated with supervising, advising, reviewing, and editing the final version of the manuscript. All the authors have read and approved the final manuscript.

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Correspondence to Pramod C. Gadad.

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Das, B.K., Knott, R.M. & Gadad, P.C. Metformin and asarone inhibit HepG2 cell proliferation in a high glucose environment by regulating AMPK and Akt signaling pathway. Futur J Pharm Sci 7, 43 (2021). https://doi.org/10.1186/s43094-021-00193-8

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  • DOI: https://doi.org/10.1186/s43094-021-00193-8

Keywords

  • Asarone
  • Metformin HCl
  • HepG2 cells
  • High glucose
  • AMPK
  • Akt