Abstract
Background/Aim: 4-methylumbelliferone (4-MU) has received considerable attention due to its potential for cancer treatment since it inhibits cell proliferation, migration and invasion. An increasing body of evidence suggests that extracellular matrix (e.g. hyaluronan) and nutrients (e.g. glucose) in the tumor microenvironment may affect cellular responses to extracellular signals. This study investigates the role of hyaluronan and glucose on 4-MU-inhibited cell proliferation in breast carcinoma cells. Materials and Methods: 4-MU-inhibited cell proliferation was determined using the soluble formazan dye, whose absorbance is directly proportional to the number of living cells, under conditions of competent vs. deficient in producing hyaluronan, or low vs. high glucose in culture media of breast carcinoma cells. Results: Cellular sensitivity to 4-MU-inhibited cell proliferation was altered by changes in the amount of hyaluronan or glucose in the tumor microenvironment. Conclusion: Increased amounts of hyaluronan or glucose in the tumor microenvironment reduced cellular sensitivity of breast carcinoma cells to 4-MU-inhibited cell proliferation.
4-Methylumbelliferone (4-MU) is a specific inhibitor of hyaluronan (HA) synthesis (1, 2). Previous studies have shown that 4-MU augments the activity of UDP-glucuronyl transferases (UGT) which catalyzes the glucuronidation of 4-MU using UDP-glucuronic acid (UDP-GlcUA) as a donor substrate (3, 4). Therefore, 4-MU inhibits HA synthesis by depleting UDP-GlcUA, one of the substrates for HA synthesis (5). In addition, 4-MU inhibits HA synthesis by reducing expression of hyaluronan synthase (HAS1, HAS2 and HAS3) (4, 5). However, 4-MU had no effects on sulfated glycosaminoglycan (GAG) synthesis in human skin fibroblasts (6, 7). A number of other studies suggest that 4-MU is a potential drug for cancer treatment since it inhibits cell proliferation, cell migration, and cell invasion in leukemia, hepatocellular carcinoma, prostate cancer, and ovarian cancer (8-12).
The tumor microenvironment (TME) consists of stromal cells, inflammatory cells, extracellular matrix (e.g. HA), nutrients (e.g. glucose), and other soluble molecules. A growing body of evidence illustrates that the TME plays an essential role in tumor initiation, development and progression (13, 14). HA is a ubiquitously expressed macromolecule located within the extracellular matrix and plays an important role in cell homeostasis, cell proliferation, and cell migration (15). HA is highly expressed in malignant tumors (e.g. breast, ovarian, and prostate cancer) and this high expression indicates a poor prognosis (16).
HA is a linear, unsulfated glycosaminoglycan composed of repeating disaccharides of glucuronic acid and N-acetyl glucosamine. HA is synthesized by HAS located on the cell membrane utilizing UDP-GlcUA and UDP-N-acetyl glucosamine (UDP-GlcNAc) as substrates for HA synthesis (15, 17). There are three HAS isoenzymes (HAS1, HAS2, HAS3) that produce variable molecular weights and amounts of HA in vertebrates (18, 19). The HAS2 isoenzyme is the major HAS in many common tumor cells (20).
In addition to the HA within the extracellular matrix, nutrients (e.g. glucose) in the TME affect various cellular responses to extracellular signals. Cancer cells demonstrate a much higher rate of glucose consumption compared to normal cells (21, 22). Cancer cells prefer utilizing glycolysis rather than oxidative phosphorylation for glucose metabolism even under oxygen-rich conditions (the Warburg effect) in order to support their rapid proliferation. Epidemiological studies reveal that the prevalence of breast cancer is high in diabetic patients and that diabetic patients are susceptible to progression of metastatic breast cancer (23-26).
4-MU Inhibition of Cell Proliferation in T-47D (A) and MDA-MB-231 (B) Human Breast Carcinoma Cells. A This graph shows that 0.25 mM 4-MU significantly (**) inhibits T-47D cell proliferation versus the untreated control (1.19 versus 1.83; p<0.01) after a 24 h treatment as measured by absorbance at 450 nm. B This graph shows that 0.25 mM 4-MU significantly (***) inhibits MDA-MB-231 cell proliferation versus the untreated control (1.71 versus 1.92; p<0.001) after a 24 h treatment as measured by absorbance at 450 nm.
In the past, the role of cellular responses to therapeutic drugs has received more attention than the role of the TME as a significant factor in cancer treatment. In the present study, we investigated the influence of two important components of the TME, namely HA and glucose, on 4-MU-inhibited cell proliferation to further explore the possibility that 4-MU may be a useful therapeutic agent for cancer treatment. Our general hypothesis is that the extracellular matrix and nutrients in the TME alter the cellular response to therapeutic drugs for cancer treatment. This study sheds light on the evaluation of the efficacy of therapeutic drugs for cancer treatment with respect to the composition of the TME.
Materials and Methods
Materials and reagents. Human breast carcinoma cell lines T-47D and MDA-MB-231 and murine breast carcinoma cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA). High-glucose Dulbecco's Modified Eagle's Medium (DMEM), low-glucose DMEM, RPMI-1640 and fetal bovine serum were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). 4-MU was purchased from Sigma (St. Louis, MO, USA). pLV-RNAi vector system was from BIOSETTLA Inc. (San Diego, CA, USA). High Molecular Weight (HMW)-hyaluronan and HA Enzyme-Linked ImmunoSorbent Assay (ELISA) kit were obtained from R&D (Minneapolis, MN, USA). Cell Counting Kit-8 was from Dojindo (Rockville, MD, USA). Anti-HAS2 was from Abgent (San Diego, CA, USA), and anti-β-actin was from Santa Cruz (Dallas, TX, USA). TransScript First-Strand cDNA Synthesis SuperMix, 2X EasyTaq PCR SuperMix and TransStart Top Green qPCR SuperMix were from TransGen Biotech (Beijing, China).
Cell culture and treatment. T-47D cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C, 5% CO2. MDA-MB-231 cells were cultured in high-glucose DMEM supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C, 5% CO2. 4T1 cells were cultured in RPMI-1640 supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C, 5% CO2.
For 4-MU-inhibited proliferation of cells, cells were plated at a density of 1×104/well of 96-well plates in the presence of different concentrations of 4-MU and cultured for 24 h, then cell proliferation was determined. HMW-HA was added or cells were switched to high-glucose medium when incubated with different concentrations of 4-MU for HA/glucose rescue experiment.
Establishment of 4T1 Has2 knockdown stable cell line. Murine Has2 shRNA sequence used was as follows: 5’-AAAAGCTGCCTTAGAGGAAATATTTGGATCCAAATATTTCCTCTAAGGCAGC-3’. This was cloned into the pLV-H1-EF1a-puro vector and transfected to 293T cells for shHas2 lentivirus production. 4T1 cells were then infected with shHas2 lentivirus and selected using 2. 5 ug/ml puromycin for 1 week to establish 4T1-shHas2 stable cell line. Murine shRNA against β-galactosidase was used as a control (4T1-sc).
Cell proliferation assay. Cell proliferation was measured using Cell Counting Kit-8 according to the manufacturer's instruction.
RT-PCR. Total RNA extracted from 4T1-sc and 4T1-shHas2 cells was first reverse-transcribed to cDNA. Then synthesized cDNA was amplified using specific murine Has2 primers. Murine Gapdh was used as a control. The PCR condition was as follows: denature at 95°C for 2 min, then with 35 cycles of denature (95°C for 1 min), annealing (60°C for 1 min) and extension (72°C for 1 min). At the end, extention at 72°C for 5 min. The conventional RT-PCR primer sequences were as follows: Has2: forward: 5’-ATTGTTGGCTACCAGTTTATCCAAAC-3’, reverse: 5’-TTTCTTTATGGGACTCTTCTGTCTCACC-3’; Gapdh: forward: 5’-CACCATGGAGAAGGCCGGGG-3’, reverse: 5’-GACGGACACATTGGGGG TAG-3’ (27). The PCR products were electrophoresed on 1.2% agarose gel and visualized using Quantity One 4.6.9 ChemiDoc XRS. The qRT-PCR primer sequences were as follows: Has2: forward: 5’-GGAACTCAGACGACGACC-3’, reverse: 5’-AAGCC ATCCAGTATCTCACG -3’; Gapdh: forward: 5’-CTCTGATTTGGTCGATTGGG-3’, reverse: 5’-TGGAAGATGG TGATGGG ATT -3’. Relative Has2 expression was obtained using Has2 values normalized against corresponding levels of Gapdh.
Establishment of a 4T1 HAS2 Knockdown Stable Cell Line (4T1-shHas2 cells). A This gel shows that the Has2 mRNA expression level is reduced in the 4T1-shHas2 cells versus the 4T1-sc cells using conventional RT-PCR. Gapdh was used as a loading control. B This graph shows that the Has2 mRNA relative expression level is significantly (***) reduced in the 4T1-shHas2 cells versus the 4T1-sc cells (0.335 versus 1; p<0.001) using quantitative Real-Time PCR. C This Western blot shows that the HAS2 protein expression level is reduced in the 4T1-shHas2 cells versus the 4T1-sc cells. β-actin was used as a loading control. D This ELISA shows that the amount of HA produced by the 4T1-shHas2 cells is significantly (*) lower than the amount of HA produced by the 4T1-sc cells (0.100 ng/ml versus 0.159 ng/ml; p<0.05).
Western blot. Protein samples isolated from 4T1-sc and 4T1-shHas2 cells were mixed with loading buffer and electrophoresed on 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Then protein samples were transferred to polyvinylidene fluoride membrane. After blocking with 5% non-fat milk, the membrane was incubated with either primary antibody to HAS2 (1:1000) or to β-actin (1:1000) overnight at 4°C. The membrane was exposed to chemiluminescence after incubation with corresponding secondary antibody to detect the specific bands.
HA ELISA. 4T1-sc and 4T1-shHas2 cells were seeded in 6-well plates at a density of 1×106/well and cultured for 48 h. Condition medium of corresponding cells was collected to determine the amount of HA using HA ELISA kit according to the manufacturer's instruction.
Statistics. Statistical analyses were performed using Student t-test (two-tailed). Results are expressed as mean±SD, with a 95% confidence interval. The results were considered statistically significant when p<0.05.
HA reduced cellular sensitivity to 4-MU-inhibited cell proliferation of 4T1 cells. A This bar graph shows that 300 μM 4-MU significantly (**) inhibits cell proliferation versus the untreated control (0.92 versus 1.07; p<0.01) in HA competent 4T1-sc cells after a 24 h treatment as measured by absorbance at 450 nm. B This bar graph shows that 100 μM 4-MU significantly (*) inhibits cell proliferation versus the untreated control (0.95 versus 1.10; p<0.05) in HA deficient 4T1-shHas2 cells after a 24 h treatment as measured by absorbance at 450 nm. C This bar graph shows that 24 h treatment of 300 μM 4-MU significantly (*) inhibits cell proliferation versus the untreated control (0.91 versus 1.09; p<0.05) in HA deficient 4T1-shHas2 cells cultured in the presence of exogenous HMW-HA as measured by absorbance at 450 nm.
Glucose reduced cellular sensitivity to 4-MU-inhibited cell proliferation of MDA-MD-231 cells. A This bar graph shows that 30 μM 4-MU significantly (**) inhibits cell proliferation versus the untreated control (1.22 versus 1.79; p<0.01) in MDA-MB-231 cells cultured in high glucose medium after a 24 h treatment as measured by absorbance at 450 nm. B This bar graph shows that 10 μM 4-MU significantly (*) inhibits cell proliferation versus the untreated control (1.20 versus 1.59; p<0.05) in MDA-MB-231 cells cultured in low glucose medium after a 24 h treatment as measured by absorbance at 450 nm. C This bar graph shows that 24 h treatment of 30 μM 4-MU significantly (*) inhibits cell proliferation versus the untreated control (1.40 versus 1.72; p<0.05) in MDA-MB-231 cells originally cultured in low glucose medium, then switched to high glucose medium as measured by absorbance at 450 nm. D This bar graph shows that 24 h treatment of 10 μM 4-MU significantly (**) inhibits cell proliferation versus the untreated control (1.20 versus 1.58; p<0.01) in MDA-MB-231 cells cultured in low glucose medium in the presence of exogenous HMW-HA as measured by absorbance at 450 nm.
Results
4-MU inhibits cell proliferation of human breast carcinoma cell lines. We examined the effect of 4-MU on cell proliferation in T-47D human breast carcinoma cells and MDA-MB-231 cells under normal culture conditions. We found that 24-h treatment of 4-MU inhibits proliferation of both T-47D cells and MDA-MB-231 cells. This inhibition is dose-dependent with a significant inhibition in cell proliferation first occurring at 0.25 mM 4-MU (Figure 1).
HA mediates cellular sensitivity to 4-MU in murine breast carcinoma cell line 4T1 cells. We examined the effect of HA in the extracellular matrix on 4-MU-inhibited cell proliferation in 4T1 cells. We first established a Has2-knockdown stable 4T1 cell line (4T1-shHas2 cells) that showed a decrease in HAS2 expression as indicated by reduced mRNA and protein levels (Figure 2A-C). Most importantly, 4T1-shHas2 cells produced a lower amount of HA compared to 4T1-sc cells (Figure 2D).
We then treated HA-competent 4T1-sc cells and HA-deficient 4T1-shHas2 cells with different concentrations of 4-MU for 24 h. The results indicate that 300 μM 4-MU was required to significantly inhibit cell proliferation in HA-competent 4T1-sc cells, whereas only 100 μM 4-MU was needed to significantly inhibit cell proliferation in HA-deficient 4T1-shHas2 cells (Figure 3A and B). With the addition of exogenous HMW-HA, the HA-deficient 4T1-shHas2 cells behaved like HA-competent 4T1-sc cells, whereby 300 μM 4-MU inhibited cell proliferation (Figure 3C). These results suggest that HA in the extracellular matrix reduces cellular sensitivity to 4-MU-induced inhibition of cell proliferation.
Glucose mediates cellular sensitivity to 4-MU in human breast carcinoma cell line MDA-MB-231. We examined the effects of 4-MU on cell proliferation of human breast carcinoma cell line MDA-MB-231 under high-glucose and low-glucose culture conditions. The results indicate that 30 μM 4-MU was needed to significantly inhibit cell proliferation of MDA-MB-231 cells under high-glucose culture conditions (Figure 4A). However, only 10 μM 4-MU is needed to significantly inhibit proliferation of MDA-MB-231 cells under low-glucose culture conditions (Figure 4B). These results suggest that glucose decreases cellular sensitivity to 4-MU-inhibited cell proliferation.
In order to confirm that glucose plays a role in regulation of cellular sensitivity to 4-MU-inhibited cell proliferation, MDA-MB-231 cells originally cultured under low-glucose conditions were switched to high-glucose conditions and then treated with different concentrations of 4-MU for 24 h. The results indicate that 30 μM 4-MU was needed to significantly inhibit proliferation of MDA-MB-231 cells switched to high glucose (Figure 4C). This suggests that the switch to high glucose reduces cellular sensitivity to 4-MU-inhibited cell proliferation.
In order to determine whether the role of glucose in 4-MU-inhibited cell proliferation can be compensated by HA, we added exogenous HMW-HA to MDA-MB-231 cells originally cultured under low-glucose conditions and then treated them with different concentrations of 4-MU for 24 h. The results indicate that 10 μM 4-MU was needed to significantly inhibit proliferation of MDA-MB-231 cells cultured in conditions with low-glucose plus exogenous HMW-HA. This means that the addition of exogenous HMW-HA did not change the cellular sensitivity of MDA-MB-231 cells to 4-MU when cultured under low-glucose conditions (Figure 4D). This suggests that the role of glucose may be more important than HA in 4-MU-inhibited cell proliferation.
Discussion
4-MU has been used in the past to promote bile discharge (i.e. as a cholagogue) and is presently in phase II clinical trials for treatment of hepatitis C and chronic hepatitis B (28, 29). Moreover, 4-MU is considered a valuable therapeutic agent to prevent cancer invasion and metastasis by specifically inhibiting HA synthesis (30).
In vivo studies indicate that 4-MU reduces both tumor growth and microvessel density, with relatively little non-specific organ toxicity (11). In vitro studies also indicate that 4-MU induces apoptosis and inhibits cell proliferation, motility, and invasion in prostate cancer, hepatocellular carcinoma, leukemia, and ovarian cancer (8, 9, 11, 12). In the present in vitro study, we showed that in addition to the above-mentioned cancer types, 4-MU also inhibits proliferation of human breast carcinoma cells (i.e. T-47D and MDA-MB-231 cells).
A growing body of evidence illustrates that the extracellular matrix, besides other components of the TME, plays an essential role in tumor initiation, development, and progression (13, 14). For example, chondroitin sulfate (a glycosaminoglycan) plays a role in promoting and regulating breast cancer progression and metastasis (31). Versican (an extracellular matrix proteoglycan) plays a role in cell proliferation, migration and invasion in leiomyocarcoma (32). In this in vitro study, we showed that HA in the ECM suppresses 4-MU-inhibited proliferation of 4T1 cells. Our results indicate that HA in the ECM plays an important role in mediating the cellular response to external stimuli. HA exists with different molecular weights in nature, with different properties. HMW-HA can be digested by hyaluronidase into different HA fragments. HMW-HA is considered an anti-inflammatory molecule and promotes cell proliferation, whereas LMW-HA is considered a pro-inflammatory molecule and inhibits cell proliferation. Therefore, it is reasonable to speculate that the combination of 4-MU treatment with changing of HMW-HA into LMW-HA in the ECM would improve the efficacy of 4-MU on cancer treatment.
Glucose is a prominent carbohydrate that undergoes a much higher rate of consumption in cancer cells compared to normal cells (21, 22). In this regard, glucose has been shown to promote cell proliferation in various types of cancer cells (e.g. pancreatic, colon, and breast) and interestingly, diabetic patients have a high prevalence of cancer perhaps due to chronically high blood glucose levels (23, 33, 34). In this study, we found that MDA-MB-231 cells were more sensitive to 4-MU under a low-glucose condition versus a high-glucose condition. This suggests that the reduced glucose levels in the TME would enhance the inhibitory effect of 4-MU on cancer cell proliferation. Although a body of evidence indicates that HA production is related to glucose levels, our results suggest that the impact of glucose on cell proliferation occurs not only through HA production but also by other pathways. In this regard, it has been reported that glucose promotes MCF-7 human breast cancer cell proliferation by acceleration of cell-cycle progression and the involvement of Protein Kinase C-α, peroxisome proliferator-activated receptor α (PPARα), and PPARγ (35).
In summary, our results indicate that low levels of HA and glucose in the TME increase the sensitivity of breast cancer cells to 4-MU, thereby inhibiting cell proliferation more dramatically. This suggests that the efficacy of various therapeutic drugs can be increased if combined with an approach that also modifies the TME.
Acknowledgements
This work was supported by the S&T plan project of Tianjin (12JCYBJC30900) and The National Natural Science Foundation of China (81301856).
- Received April 30, 2015.
- Revision received June 7, 2015.
- Accepted June 9, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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