GLUT5, GLUT7, and GLUT11 expression and Bcl-2/Bax ratio on Breast Cancer Cell Line MCF-7 Treated with Fructose and Glucose

Objective: Fructose and glucose are types of sugars commonly found in the diet that have been linked to cancer development. Glucose transporters (GLUTs) are facilitating the uptake of these hexoses. Expression of GLUT5 is higher in cancer cells than in healthy tissue. GLUT7 and GLUT11 facilitate the transport of glucose and fructose; however, their expression in breast cancer has not been extensively studied. The Bcl-2 family has been known as a regulator of the cell’s survival and death. Here, we investigated the effect of the fructose-glucose combination in MCF-7 breast cancer cells on the viability, migration, and expression of GLUT5, GLUT7, GLUT11, and Bcl-2/Bax ratio. Methods: Breast cancer cells MCF-7 were treated with fructose, glucose, and combinations of fructose:glucose (75%:25%, 50%:50%, 25%:75%). Cell viability was assessed using an MTT test. Cell migration was examined with a wound-healing assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the mRNA expression of GLUT5, GLUT7, GLUT11, and Bcl-2/Bax. Results: The viability and migration of MCF-7 breast cancer cells elevated when treated with a combination of fructose and glucose, and glucose alone, compared to fructose alone. The expression levels of GLUT5 and GLUT7 were highest in combination of fructose:glucose (75%:25%). Conversely, the expression of GLUT11 was consistently low across all treated media. The highest Bcl-2/Bax ratio was shown in fructose:glucose combination (25%:75%). Conclusion: The viability, migration, and Bcl-2/Bax ratio are enhanced in the combination media with higher glucose. In contrast, when the fructose composition was higher in the media, expression of GLUT5 and GLUT7 increased.


Introduction
One of the cancer cell's hallmarks is altered in metabolic regulation (Schiliro and Firestein, 2021).Cancer cells can adapt through complex metabolism related to genetic factors and the nutrients in the microenvironment (Elia and Haigis, 2021;Grasmann et al., 2021).Cancer cells consume high amounts of glucose to maintain cell proliferation (Jiang, 2017).
Apart from D-glucose, D-fructose is also a critical monosaccharide for the growth of cancer cells (Nakagawa et al., 2020).Foods high in fructose can cause increase the risk of several types of cancer and tumor progression (Charrez et al., 2015;Das, 2015;Hsieh et al., 2017;Weng et al., 2018).Fructose metabolism in cancer cells contributes to the Warburg effect by increasing glycolysis RESEARCH ARTICLE GLUT5, GLUT7, and GLUT11 expression and Bcl-2/Bax ratio on Breast Cancer Cell Line MCF-7 Treated with Fructose and Glucose through the production of lactic acid (Nakagawa et al., 2021), which also stimulates the development and metastasis of breast cancer (Jiang et al., 2016).Substituting glucose with fructose as the energy source in MDA-MB-468 breast cancer cells induced an aggressive phenotype characterized by enhanced migration and invasion capabilities (Monzavi et al., 2010).Another study revealed that high fructose concentrations contribute to the advancement of colorectal cancer growth (Shen et al., 2022).
Glucose and fructose are transported into the cell by glucose transporters (GLUTs) (Shin and Koo, 2021).Cancer cells show high expressions of GLUT5, which is the main fructose transporter and also related to breast cancer prognosis (Koltai and Fliegel, 2023;Rana et al., 2023).GLUT7, on the other hand, is still controversial about the ability of which sugars it transports.Ebert et al., 2017 reported that GLUT7 neither transports fructose nor glucose.Another study showed that GLUT7 levels increased when fructose was applied to the Caco-2/ TC-7 cell monolayer (Gauer et al., 2018).Another glucose transporter, GLUT11 has the highest similarity to fructose transporter GLUT5 (42% sequence homology), and facilitates the transport of both fructose and glucose (Navale and Paranjape, 2016;Holman, 2020).Since fructose and glucose play a significant role in the progression of cancer cells, GLUTs expression should be investigated further.
Despite its role in promoting the Warburg effect and sustaining the viability of cancer cells, fructose exposure has been found to induce apoptosis in non-cancerous cells (Jaiswal et al., 2015).Therefore, it is important to conduct a further examination of fructose's contribution to the apoptosis process.The B-cell lymphoma 2 (Bcl-2) family is known as regulators of survival and cell death divided into two groups, which are pro-apoptotic and anti-apoptotic (Adams and Cory, 2018).An increase in the Bcl-2/Bax ratio is associated with resistance to paclitaxel (Sharifi et al., 2014).This study aimed to investigate the viability, migration, and expression of these transporters and their impact on the Bcl-2/Bax ratio in MCF-7 breast cancer cells treated with fructose and glucose alone or in combination.

Cell viability test
MTT assay evaluated cell viability after 24, 48, 72, and 96 hr.Briefly, cells were seeded at 5 x 103 in each well into a 96-well plate, and test media with fructose or glucose alone and combined were treated with three replicates.After the incubation, the medium was discarded and washed, then 20 µl MTT was added and incubated at 37°C for four hours.After solubilizing with 200 µl SDS, the absorbance was measured at 570 nm using a microplate reader.

Wound healing assay
Cells were seeded onto 24-well plates with a 5 x 104 cells/well density.After cells reached 80-90% confluence, a scratch was made with a 20-200 µl sterile pipette tip, generating approximately 1 mm width of cell-free area.After removing the cellular debris by gently washing it with PBS, pictures were taken (0 hr).Then, the culture media was replaced with the medium containing fructose and glucose, alone or in combination.At 6 hr postwounding, an inverted microscope was utilized to capture post-treatment pictures (five independent, random fields).
ImageJ software was used to analyze and calculate the gap size.

RNA extraction and quantitative polymerase chain reaction (qPCR)
Total RNA was isolated from cells using TRIzol reagent according to the manufacturer's instructions.RNA concentration was determined with a spectrophotometer (OD: 260nm) to synthesize cDNA with an iScriptTM cDNA synthesis kit (Biorad).The primer sequences are listed in Table 1, with ß-actin used as the reference gene.qPCR was performed in 10 µl reaction volume, with 5 µl 2X SensiFAST SYBR Green PCR master mix and 1 µl of specific primer set.The qPCR was performed at 95oC for 15 min, followed by 40 cycles of 94oC for 15 sec, 53oC for 30 sec, and 72oC for 30 sec.Each sample was examined in triplicate.Relative gene expression was calculated by the Livak method (Livak and Schmittgen, 2001).

Statistical analysis
All quantitative data are presented as mean ± standard deviation (SD) from three independent experiments.Significant difference among the group was assessed by One-way analysis of variance (ANOVA) and post hoc Tukey's test, with P<0.05 considered statistically significant.

More glucose in media resulted in enhanced MCF7 viability
Viability cell was measured at 24, 48, 72, and 96 hr after cultured in media supplemented with fructose alone, glucose alone, or combination of both hexoses.The results showed that cell viability was greater in cultures treated with a combination of both hexoses compared to those treated with fructose alone.
The results shown in Figure 1, after 24, 48, and 72 hr, demonstrated a similar pattern, cells treated with glucose alone had the highest cell viability.The treatment with 50:50 of fructose and glucose exhibited the highest levels after 96-hour treatment.However, after incubation at all times, cells treated with fructose alone retained the lowest viability.

Fructose-glucose combination affected cell migration
Migration assay was analyzed by measuring the percent wound closure using ImageJ software (Figure 2).Cells cultured with fructose and glucose alone or combined media had a significantly higher percentage of wound closure than the control group.However, the combination media with an equal or higher glucose content (fructose:glucose 50%:50%, 25%:75%) and glucose alone had a significantly higher percentage of wound closure than the combination media with a higher fructose content and fructose alone.This result represented a higher The greatest fold change for GLUT5 and GLUT7, as shown in Figure 3 was obtained at combination fructose:glucose (75%:25%).Meanwhile, GLUT11 showed the lowest expression in all experiment media.In media with fructose alone, the mRNA expression of GLUT5, GLUT7, and GLUT11 was lower than in the control medium without glucose and fructose.The same pattern was also shown in the culture with media combining fructose:glucose 25%:75%.In media with glucose alone, GLUT5 had a slight increase in expression but was not applied for GLUT7 and GLUT11.Interestingly, in the combination media fructose:glucose migration rate observed when the cells were exposed to a higher concentration of glucose than an excessive concentration of fructose in the media.After 24 hr, the gap in all cell culture experiments was closed, except control without fructose and glucose.

GLUT level and Bcl-2/Bax ratio
The cell was cultured with fructose, glucose, and a combination of both for 72hr, and mRNA levels of GLUT5, GLUT7, and GLUT11 were measured by RT-qPCR.Data was calculated and represented by relative quantitative.
Finally, the expression of Bcl-2 and Bax was measured with RT-qPCR, as presented in Figure 4.For Bcl-2, cells treated with 100% glucose showed the highest fold change (~2).Wherewith Bax, the expression displayed all below to the control.For the Bcl-2/Bax ratio, the combination of fructose:glucose (25%:75%) has the highest ratio.The 100% fructose group and the combination of fructose: glucose (75%:25%) showed a lower Bcl-2 and Bax expression than the control group, as did the ratio values.

Discussion
In this study, we have shown that the combination of fructose and glucose affected the viability and migration of breast cancer cells.The higher the percentage of glucose in the combination media, the greater the cell viability.Expression of GLUT5, GLUT7, GLUT11 This result confirmed that breast cancer cells, especially MCF7, depend on glucose as their glycolytic substrate to support viability.It was also shown that when these cells were grown in media with glucose alone, they consistently had the highest viability compared to other media groups.
Meanwhile, cells grown in media with fructose alone had viability that was not significantly different from control media (without glucose and fructose).The last result was different from the results of a previous study (Fan et al., 2017) on MFC7 and MDA-MB, which found that the survival of these two cells cultured with 10 mM fructose-supplemented media was almost similar to 25 mM glucose-supplemented media and significantly higher compared to culture without glucose.However, another study on Jurkat cells (a lymphoblastic leukemia cell model) showed that 10 mM fructose induced cell death through elevated production of superoxide and hydrogen peroxide due to increased fructose oxidation through the oxidative phosphorylation pathway (Diaz et al., 2016).So, further studies are needed to explain at what levels of fructose inhibit cell viability or induce apoptosis or, conversely, strengthen cancer cells' survival by inhibiting the tricarboxylic acid cycle, which preserves the Warburg effect.The migration capacity of breast cancer cells in this study, as represented by the percentage of wound closure after 6 hours, showed results that aligned with the cell viability.
Glucose transporters have unique monosaccharide sensing and response abilities, as well as their cell-specific expression.Studies have indicated that breast cancer cells exhibit overexpression of glucose transporters, including GLUT1, GLUT3, GLUT4, and GLUT12, which play crucial roles in metabolism and the uptake of hexose molecules (McBrayer et al., 2012;Reinicke et al., 2012).A previous study indicated that GLUT5 is not over-expressed in breast cancer cells or tissue (Gowrishankar et al., 2011).In a recent study, the participation of GLUT-2 as a fructose transporter in breast cancer was discovered (Hamann et al., 2018).The current study observed that MCF7 cells showed a higher GLUT5 expression pattern compared to GLUT7 and GLUT11 when cultured in fructose and glucose alone, and in combination media with a greater percentage of glucose than fructose.However, the expression levels of these three genes were lower than the control.These results indicate that GLUT5, GLUT7, and GLUT11 are not the primary transporters for fructose and glucose in breast cancer cells.However, at a percentage of fructose greater than glucose (75%; 25%), there was an increase in GLUT5 and GLUT7 mRNA expression, with GLUT7 being higher than GLUT5.These results can be initial data that GLUT5 and GLUT7 most likely play a role in transporting glucose and fructose into breast cancer cells at certain concentration combinations of these two hexoses.This notion requires further proof with more advanced techniques because this study did not measure other monosaccharide transporters, such as GLUT1, GLUT4, or GLUT12, the main glucose transporters in breast cancer cells (Laudanski et al., 2004;Garrido et al., 2015;Matsui et al., 2017).
Bcl-2 family plays a role in mitochondrial-dependent apoptosis (Bata and Cosford, 2021;Changizi et al., 2021).The Bcl-2/Bax regulates apoptosis mediated by the mitochondrial pathway (Wang et al., 2016).Our results showed that the cell viability was significantly higher when exposed to a higher glucose concentration in conjunction with the increase of the Bcl-2/Bax ratio.Meanwhile, MCF7 cultured with fructose and combination media with higher fructose content had low Bcl-2 and Bax mRNA expression.These results can be explained by a study in muscle cells showing that fructose exposure induced oxidative stress, triggered mitochondrial dysfunction, and disrupted the expression of Bcl-2 family members and caspases (Jaiswal et al., 2015).Likewise, a study on Huh7 and A52 hepatocellular carcinoma cells showed that fructose promoted apoptosis, which was most likely caused by ROS accumulation and energy insufficiency (Dewdney et al., 2020).Further studies are needed to clarify whether this mechanism also applies to other cancer cells, including breast cancer cells.
In conclusion, this study added to the evidence that the higher the portion of glucose in the media, the greater the growth and migration ability of breast cancer cells.Meanwhile, fructose without the presence of glucose only slightly increased cancer cell viability, accompanied by suppressing Bcl-2 expression.GLUT5 was most likely the main transporter for glucose and fructose, and GLUT7 was an alternative for fructose when these two hexoses were present together in the medium at a particular concentration.This research gave rise to unexpected results regarding the role of fructose in cell viability and growth and its capability to induce apoptosis of breast cancer cells.Therefore, further studies are needed to clarify the role of fructose, at what levels can this hexose alone support the growth and increase the progression of breast cancer cells or, conversely, have the potential to suppress the cells' growth.

Figure 1 .
Figure 1.Effect of Various Combinations of Fructose and Glucose Supplemented in Growth Media on MCF-7 viability after 24, 48, 72, and 96 hr with MTT Assay.Viable cells percentage was calculated as follows: (absorbance of test media cell -absorbance of control media) / (absorbance of control cell -absorbance of control media) x 100%.Results are presented as mean ± SD (n=3).The stars indicate a significant difference compared with cells treated without fructose and glucose.****P<0.001;F, Fructose; G, Glucose

Figure 3 .
Figure 3. Relative mRNA Expression of GLUT5, GLUT7, and GLUT11 for Each Experiment.Relative mRNA expression calculated with 2ΔΔCT method.Data were expressed as mean ± SD from three independent test.Controls are shown with dotted lines.F, Fructose; G, Glucose Figure 4. Expression of Bcl-2, Bax and Ratio of Bcl-2/Bax in Breast Cancer Cell MCF-7 Treated with Various Combinations of Fructose and Glucose.F, Fructose; G, Glucose