Edited by: Olivier Cuvillier, Centre National de la Recherche Scientifique (CNRS), France
Reviewed by: Claire Perks, University of Bristol, United Kingdom; Timothy LaRocca, Albany College of Pharmacy and Health Sciences, United States
This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Pharmacology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Diabetes and cancer are common, chronic, and potentially fatal diseases that frequently co-exist. Observational studies clearly indicate that the risk of several types of cancer is increased in diabetic patients and a number of cancer types have shown a higher mortality rate in patients with hyperglycemic associated pathologies. This scenario could be due, at least in part, to a lower efficacy of the cancer treatments which needs to be better investigated. Here, we evaluated the effects of a prolonged exposure to high glucose (HG) to the response to chemotherapy on human colon adenocarcinoma HT29 and LOVO cell lines. We observed that hyperglycemia protected against the decreased cell viability and cytotoxicity and preserved from the mitochondrial DNA lesions induced by doxorubicin (DOX) and 5-fluorouracil (5-FU) treatments by lowering ROS production. In HT29 cells the amount of intracellular DOX and its nuclear localization were not modified by HG incubation in terms of Pgp, BCRP, MRP1, 5 and 8 activity and gene expression. On the contrary, in LOVO cells, the amount of intracellular DOX was significantly decreased after a bolus of DOX in HG condition and the expression and activity of MPR1 was increased, suggesting that HG promotes drug chemoresistance in both HT29 and LOVO cells, but in a different way. In both cell types, HG condition prevented the susceptibility to apoptosis by decreasing the ratio Bax/Bcl-2 and Bax/Bcl-XL and diminished the level of cytosolic cytochrome c and the cleavage of full length of PARP induced by DOX and 5-FU. Finally, hyperglycemia reduced cell death by decreasing the cell percentage in sub-G1 peak induced by DOX (via a cell cycle arrest in the G2/M phase) and 5-FU (via a cell cycle arrest in the S phase) in HT29 and LOVO cells. Taken together, our data showed that a prolonged exposure to HG protects human colon adenocarcinoma cells from the cytotoxic effects of two widely used chemotherapeutic drugs, impairing the effectiveness of the chemotherapy itself.
Antineoplastic drugs are an important therapeutic tool for cancer patients, but the development of multidrug resistance (MDR) may result in failure of the treatment, leading to tumor relapse and further progression. One of the mechanisms of MDR is the overexpression of the ATP binding cassette (ABC) transporters, such as P-glycoprotein (Pgp) and other MDR-related proteins (e.g., BCRP and MRPs), that actively extrude anticancer drugs, such as anthracyclines (e.g., doxorubicin, DOX), taxanes, Vinca alkaloids, epipodophyllotoxins, topotecan, mitomycin C (
The most recent International Diabetes Federation’s estimates indicate that 12.8% of adults – 382 million people worldwide in 2016 – have diabetes, and the number of people with the disease is set to rise beyond 592 million in less than 25 years, whereas 1.5 million people die from this disease every year (
Hyperglycemia is a pathophysiological condition characterized by high blood glucose concentration that is not only a key pathological factor involved in diabetic complications (
Even if several signaling pathways have been evaluated with respect to their involvement in drug resistance and hyperglycemia, gaining a better understanding of the mechanisms underlying the failure of cancer treatment may improve the drug efficacy in this condition.
In the present study, we evaluate the effects of a prolonged exposure to high glucose (HG) in the response to two chemotherapeutic drugs [DOX and 5-fluorouracil (5-FU)] on human colon adenocarcinoma (HT29 and LOVO cell lines).
Human colon adenocarcinoma (HT29 and LOVO cell lines) obtained from the American Type Culture Collection (Rockville, MD, United States) were grown as a subconfluent monolayer in DMEM and F12/DMEM medium, respectively, containing 2 mM
Unless otherwise specified, reagents were purchased from Sigma-Aldrich (Milan, Italy), whereas plastic ware was from Falcon (Becton Dickinson, Franklin Lakes, NJ, United States).
Different types of cells were cultured up to 12 weeks in medium containing normal glucose (control cells, G, 5.56 mM equal to 100 mg/dL) or HG (25 mM equal to 450 mg/dL). Mannitol (M, 25 mM) was used as osmotic control cells. Glucose concentrations of 5.56 mM and 25 mM, chosen in this study, correspond to glycemia in fasting healthy individuals and to hyperglycemia encountered in diabetic patients poorly controlled.
As preliminary experiments, we evaluated one-weekly lactate dehydrogenase (LDH) leakage and DOX intracellular accumulation up to 12 weeks in HG condition (data not shown). Given the similar results observed after 1 week and after the following experimental points, the experimental conditions have been standardized using cells cultured in very HG not less than 7 days (HG ≥ 7 days).
When required, cells were incubated for 24, 48, or 72 h before analysis with different doses of DOX or of 5-FU. 5-FU was chosen as one of the main elective drugs for colorectal cancer treatment, whereas DOX, used as drug for solid tumors, was also easily detectable in our experimental approaches.
Cells were grown in G and HG ≥ 7 days, treated for 24, 48, or 72 h, respectively, with chemotherapeutic agents at scalar concentrations (from 0.05 μmol/L to 10 μmol/L for DOX or from 1 μmol/L to 50 μmol/L for 5-FU, then stained for 1 h at 37°C in culture medium containing Neutral Red solution, washed three times with phosphate-buffered saline solution (PBS) and rinsed with stop buffer (1:1 of 4.02 g trisodium citrate in 153 mL H2O, 0.8 mL HCl 0.1 N in 86 mL H2O and 25 ml of 95% v/v methanol), as described (
For LDH leakage measurement, cells were grown in G and HG ≥ 7 days, then after 24 and 48 h of incubation with DOX (from 0.5 μmol/L to 10 μmol/L) or after 72 h of 5-FU (25 and 50 μmol/L), the extracellular medium was withdrawn and centrifuged at 12,000 ×
Cells were grown in G and HG ≥ 7 days, then, after 24 h incubation in the absence or presence of 5 and 10 μM DOX or 25 and 50 μM 5-FU, cells were divided into two aliquots: one aliquot, to detect total cellular ROS production, has been resuspended in fresh appropriate medium; the other one, to detect mitochondrial ROS production, has been used to prepare mitochondrial fraction (see details in “Materials and Methods,” section “Cytochrome c Western Blot Analysis”). Total and mitochondrial lysates were loaded for 15 min with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is a cell-permeable probe that is cleaved intracellularly by non-specific esterases to form DCFH, which is further oxidized by ROS to form the fluorescent compound dichlorofluorescein (DCF). After the incubation, cells were washed twice with PBS to remove excess probe, and total and mitochondrial DCF fluorescence were determined at an excitation wavelength of 504 nm and an emission wavelength of 529 nm, using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT, United States). The fluorescence value was normalized to the protein content and expressed as RFU/mg cellular proteins or mitochondrial proteins (
Cells were grown in G and HG ≥ 7 days, then incubated with DOX (5 and 10 μM for 24 h) or 5-FU (25 and 50 μM for 72 h) and washed with ice-cold PBS twice. Total DNA was purified using the Extract-NAmpTM polymerase chain reactions from tissue kit containing all the reagents needed to rapidly extract and amplify human genomic DNA. Briefly, 10 μl of cells was mixed with 20 μl of the extraction solution and the mixture was incubated at room temperature for 5 min. After adding 180 μl of the neutralization solution, the extract was ready for determine the mtDNA integrity by qRT-PCR and the lesion rate by semi-long run rt-PCR (SLR rt-PCR).
The qRT-PCR was carried out as described by
The SLR rt-PCR amplifications were conducted in according to
To compare the levels of DNA lesion in each tested region of the mitochondrial genome, two mtDNA fragments of different lengths [long fragments (L) ranging from 972 to 1037 bp and small fragments (S) from 54 to 87 bp, respectively], located in the same mitochondrial genomic region were used. To quantify the mitochondrial lesion frequency the four 1 kb sized mtDNA regions selected for the SLR rt-PCR approach were the following [(a) D-loop region, chrM:16021+423, (b) ATPase region, chrM:8204+9203, (c) ND4/5 region, chrM:12050+13049, (d) ND1/2 region, chrM:3962+4998]. The specific oligonucleotides were: for short amplicon AS1.F/R (5′-CCCTAACACCAGCCTAACCA-3′ and 3′-AAAGTGCATACCGCCAAAAG-5′), BS1.F/R (5′-CATGCCCATCGTCCTAGAAT-3′ and: 3′-ACGGGCCCTATTTCAAAGAT-5′), CS1.F/R (5′-TCCAACTCATGAGACCCACA-3′ and 3′-TGAGGCTTGGATTAGCGTTT-5′), DS1.F/R (5′-ACTACAACCCTTCGCTGACG-3′ and 3′-GCGGTGATGTAGAGGGTGAT-5′) and for long amplicon AL4.F/AS1.R (5′-CTGTTCTTTCATGGGGAAGC-3′ and 3′-AAAGTGCATACCGCCAAAAG-5′), BL1.F/R (5′-CATGCCCATCGTCCTAGAAT-3′ and 3′-TGTTGTCGTGCAGGTAGAGG-5′), CL1.F/R (5′-CACACGAGAAAACACCCTCA-3′ and 3′-CTATGGCTGAGGGGAGTCAG-5′), DL1.F/R (5′-CCCTTCGCCCTATTCTTCAT-3′ and 3′-GCGTAGCTGGGTTTGGTTTA-5′). The reaction mix consisted of 1 μl iTaqTM Universal SYBR® Green Supermix, 500 nM each forward and reverse primer (specific for the long and the short amplicon) and the equivalent quantities of template DNA (3 ng of total DNA) in 10 μl total volume. The cycling conditions include a pre-incubation phase of 10 min at 95°C followed by 40 cycles of 10 s 95°C, 10 s 60°C, and 10 s 72°C (for small fragments) or 50 s 72°C (for large fragments). Each sample was assayed in triplicate, fluorescence was continuously monitored versus cycle numbers and crossing point values were calculated by the CFX96 TouchTM software (Bio-Rad). The PCR conditions for the different fragments were optimized to achieve similar amplification efficiencies required to compare different amplicons. The product specificity was monitored by melting curve analysis and product size was visualized on agarose gel by electrophoresis (data not shown).
For the quantification of damage in each mtDNA region, the crossing point (Cp) of isolated mitochondrial DNA from untreated sample was taken as reference. For each of the four mtDNA regions the difference in the crossing point ΔCp (long fragment/small fragment) was used as a measure of the relative mitochondrial lesion frequency with the 2-ΔΔCT method in correlation to the amplification size of the long fragment and expressed as lesion per 10 kb DNA of each mtDNA region by including the size of the respective long fragment and displayed as average of at least three independent experiments (
Lesion rate [Lesion per 10 kb DNA] = (1–2 - (Δ long - Δ short)) × 10000 [bp]/size of long fragment [bp].
Before every test, cells were grown in G and HG ≥ 7 days and incubated with DOX (5 and 10 μM for 24 h or 0.5 and 1 μM for 48 h), then washed twice in PBS, and cells were scrapered and collected. Cells were centrifuged for 30 s at 13,000 rpm (4°C) and resuspended in 700 μl of a 1:1 mixture of ethanol/0.3 N HCl. Fifty microliters of cell suspension were sonicated on crushed ice with two 10-s bursts (Labsonic sonicator, 100 W) and used for measurement of cellular proteins; the remaining part was checked for the DOX content using a Perkin-Elmer LS-5 spectrofluorimeter (Perkin Elmer). Excitation and emission wavelengths were 475 and 553 nm, respectively. A blank was prepared in the absence of cells in every set of experiments and its fluorescence was subtracted from that obtained in the presence of cells (
Cells were grown in G and HG ≥ 7 days and then incubated with DOX (5 and 10 μM for 24 h) washed twice in ice-cold PBS and resuspended in 500 μl of PBS. Cellular samples derived from DOX incubation were diluted 1:1 with acetonitrile 0.1% HCOOH; the mixture was sonicated, centrifuged for 10 min at 2150 g, filtered (0.45 μm PTFE) and analyzed by RP-HPLC. HPLC analyses were performed with a HP 1200 chromatograph system (Agilent Technologies, Palo Alto, CA, United States) equipped with a quaternary pump (model G1311A), a membrane degasser (G1322A), a multiple wavelength UV-vis detector (MWD, model G1365D), a thermostated column compartment (model G1316A), and fluorescence detector (FLD, model G1321A) integrated in the HP1200 system. Data analysis was performed using a HP ChemStation system (Agilent Technologies). Samples were analyzed according to
The micromolar concentration value was normalized to the protein content and expressed as nmol DOX or metabolites/mg cell proteins.
Cellular samples derived from 5-FU incubation (25 and 50 μM for 24, 48, and 72 h) were added with acetonitrile (4:1, v/v); the mixture was sonicated, centrifuged for 10 min at 2150 ×
5-FU content in cellular culture medium was assessed after 72 h incubation in the presence of HT29 and LOVO cells. Supernatant was diluted 1:1 with acetonitrile; the mixture was sonicated, centrifuged for 10 min at 2150 ×
Samples were analyzed according to
0.5 × 106 cells, incubated in the experimental conditions reported previously, were grown on sterile glass coverslips, rinsed with PBS, fixed with 4% w/v paraformaldehyde (diluted in PBS) for 15 min, washed three times with PBS and incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, diluted 1: 20000) for 3 min at room temperature in the dark. Fluorescently labeled cells were washed three times with PBS and once with water, then the slides were mounted with 4 μl of Gel Mount Aqueous Mounting and examined with a Leica DC100 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). For each experimental point, a minimum of five microscopic fields were examined.
The efflux of rhodamine 123, a substrate of Pgp and MRP, was taken as an index of Pgp plus MRP activity. Cells were grown in G and HG ≥ 7 days, then washed with fresh PBS, detached with cell dissociation solution and resuspended at 5 × 105 cells/mL in 1 mL of DMEM medium containing 5% FBS. The samples were maintained at 37°C for 20 min in the presence of 1 μg/mL rhodamine 123. After this incubation time, cells were washed and resuspended in 500 μl of PBS, and the intracellular rhodamine content, which is inversely related to its efflux, was analyzed for the rhodamine content, using a PerkinElmer LS-5 spectrofluorimeter. Excitation and emission wavelengths were 507 and 527 nm, respectively. An aliquot of sample was used for the determination of the intracellular proteins. A blank was prepared in the absence of cells in each set of experiments, and its fluorescence was subtracted from the one measured in each sample. Fluorescence was converted in % of rhodamine 123/mg of cell proteins using a calibration curve prepared previously.
The efflux of Hoechst 33342, a specific substrate of BCRP, was taken as an index of BCRP activity. Cells were grown in G and HG ≥ 7 days, then washed with PBS and resuspended in 500 μL of DPBS buffer (129 mM NaCl, 2.5 mM KCl, 7.4 mM Na2HPO4, 1.3 mM KH2PO4, 1 mM CaCl2, 0.7 mM MgSO4, 5.3 mM glucose; pH 7.4), in the presence of 50 μM Hoechst 33342 for 15 min at 37°C. Then 400 μl of stop solution (210 mM KCl, 2 mM Hepes; pH 7.4) was added and cells were lysed with 100 μl of 0.1% v/v Triton-X 100, dissolved in 0.3% v/v NaOH.
An aliquot of sample was used for the determination of the intracellular proteins, and the remaining part was analyzed for the Hoechst content, using a PerkinElmer LS-5 spectrofluorimeter. Excitation and emission wavelengths were 370 and 450 nm, respectively. A blank was prepared in the absence of cells in each set of experiments, and its fluorescence was subtracted from the one measured in each sample. Fluorescence was converted in % of Hoechst/mg of cell proteins using a calibration curve prepared previously.
Before every test, cells were grown in G and HG ≥ 7 days and then washed with PBS. Total RNA was extracted with TRIzol® (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States). One μg of total RNA were reversely transcribed into cDNA, in a final volume of 20 μl, using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, United States) according to the manufacturer’s instructions. The RT-PCR primers were designed with NCBI/Primer-BLAST. Quantitative PCR was carried out in a final volume of 20 μl using the iTaqTM Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, United States) with specific primers for the quantitation of
PCR amplification was 1 cycle of denaturation at 95°C for 30 s, 40 cycles of amplification including denaturation at 95°C for 30 s and annealing/extension at 60°C for 1 min. Standard curves, with serially diluted solutions (1:1; 1:10; 1:100; and 1:1000 for MRP1/5/8 and BCRP genes and 1:1; 1:10; 1:100 for Pgp) of cDNAs obtained as a template for each gene, were included in each PCR and amplified by target-specific primer sequence to quantify the PCR baseline subtracted relative fluorescence unit. The threshold cycle (Ct) reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold line. The quantification of each sample was performed comparing each PCR gene product with B2M, used as reference gene to normalize the cDNA in different samples, and expressed in arbitrary units, using the Bio-Rad Software Gene Expression Quantitation (Bio-Rad Laboratories), calculated using the 2-ΔΔCT method (
Cells were grown in G and HG ≥ 7 days and then incubated with DOX (5 and 10 μM for 24 h) or 5-FU (25 and 50 μM for 72 h). Cells were collected and washed twice in PBS, then lysed for 1 h in ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, pH 7.4 supplemented with the protease inhibitor cocktail set III (Sigma-Aldrich, Milan, Italy), 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 1 mg/ml aprotinin, 50 mM sodium fluoride, 1% Triton X-100). Cell lysates were then centrifuged for 15 min at 13,000 rpm at 4°C (
Cells were grown in G and HG ≥ 7 days and then incubated with DOX (5 and 10 μM for 24 h) or 5-FU (25 and 50 μM for 72 h). The cell supernatant was collected, centrifuged at 4,000 rpm for 2 min at 4°C and, after washing with ice-cold PBS twice, resuspended in mitochondrial lysis buffer A (50 mM Tris, 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, ATP 1.8 mM, pH = 7.2). 5 × 106 cells were washed twice in ice-cold PBS, then added to the pellet derived from cell supernatant and resuspended in 500 μl of mitochondrial lysis buffer A, supplemented with the protease inhibitor cocktail set III (Sigma-Aldrich, Milan, Italy), 1 mM phenylmethylsulfonyl fluoride and 2.5 mM sodium fluoride. Samples were clarified by centrifuging at 2,000 rpm for 2 min at 4°C, and the supernatant was collected and centrifuged at 13,000 rpm for 5 min at 4°C. The supernatant (cytosolic fraction) was aliquoted and the pellet containing mitochondria (mitochondrial fraction) was washed in 500 μl buffer A and resuspended in 250 μl mitochondrial resuspension buffer B (250 mM sucrose, 15 mM K2HPO4, 2 mM MgCl2, 0.5 mM EDTA, 5% w/v BSA). The aliquot of mitochondrial fraction was sonicated, used for the measurement of protein concentration and stored at -80°C until the use. Ten micrograms from mitochondrial extracts were subjected to 15% SDS-PAGE, transferred to PVDF membrane and probed with the following antibodies: mouse anti-cytochrome c antibody (diluted 1:250 in PBS-BSA 1%, from BD Biosciences, San Jose, CA, United States) and mouse anti-β-tubulin (diluted 1:500 in PBS-BSA 1% from Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States). The latter antibody was used to check the equal protein loading in mitochondrial extracts. After an overnight incubation, the membrane was washed with 0.1% v/v PBS-Tween and subjected for 1 h to a peroxidase-conjugated anti-mouse secondary antibody (diluted 1:5000 in 5% w/v PBS-Tween with milk, Bio-Rad Laboratories, Hercules, CA, United States). The membrane was washed again with PBS-Tween, and proteins were detected and quantified by ChemiDocTM MP System (Bio-Rad Laboratories, Hercules, CA, United States). Densitometric analysis was carried out using ImageJ software (see footnote 2).
Cells were grown in G and HG ≥ 7 days, then, after 24 h incubation in the absence or presence of 5 and 10 μM DOX or after 72 h with 25 and 50 μM 5-FU, cells were collected, fixed in 70% cold ethanol for 30 min on ice and centrifuged at 1,800 rpm for 10 min. Cells were washed with PBS, then 1 × 106 cells/ml were resuspended in 1 ml PBS and incubated in propidium iodide solution (20 μg/ml propidium iodide and 0.2 mg/ml RNAseA in PBS) for 1 h at room temperature. Cellular DNA content was analyzed by Coulter EPICS XL (CoulterCorp, Hialeah, FL, United States) flow cytometry and to perform the analysis only on intact single cells, an electronic gate for doublet and clump exclusion was used (
The
Cells were grown in G and HG ≥ 7 days, then incubated with DOX 5 and 10 μM for 24 h and washed with ice-cold PBS twice. The cells were scraped up in PBS, collected, centrifuged at 800 ×
Data were expressed as mean ± SEM of the mean. The results were checked for normal distribution and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test comparing samples treated with the same dose of chemotherapeutic agent in HG vs. G condition. Statistical significance level was set at
The cell viability, measured by neutral red uptake assay, was significantly higher in HT29 and LOVO cells cultured in HG compared with control cells at the same doses and time incubation of DOX (
Effect of normal glucose (G) and high glucose (HG) on cell proliferation and on LDH release from cells in the supernatant in absence or presence of doxorubicin (DOX) in human colon cancer (HT29 and LOVO) cells. Cells were cultured for ≥7 days in the presence of G and HG, then subjected to the following investigations.
Effect of normal glucose (G) and high glucose (HG) on cell proliferation and on LDH release from cells in the supernatant in absence or presence of 5-fluorouracil (5-FU) in human colon cancer (HT29 and LOVO) cells. Cells were cultured for ≥7 days in the presence of G and HG, then subjected to the following investigations.
As anticancer drugs, DOX (
Effect of normal glucose (G) and high glucose (HG) on mitochondrial ROS production in absence or presence of DOX
The integrity maintaining of the mitochondrial genome (mtDNA) is a prerequisite for proper mitochondrial function (
Effect of normal glucose (G) and high glucose (HG) on mitochondrial DNA damage (mtDNA) in absence or presence of DOX in human colon cancer (HT29 and LOVO) cells. Cells were cultured for ≥7 days in the presence of G and HG, incubated for 24 h before analysis with 5 and 10 μM DOX, then washed and processed to determine the mtDNA integrity by qRT-PCR and the lesion rate by semi-long run rt-PCR (SLR rt-PCR). Measurements (
We then analyzed the effect of hyperglycemic condition on the expression and activity of the ABC transporters. In HT29 cells cultured in G or HG the amount of intracellular DOX (
Effect of normal glucose (G) and high glucose (HG) on intracellular accumulation of DOX in human colon cancer (HT29 and LOVO) cells. Cells were cultured for ≥7 days in the presence of G and HG and incubated with 5 and 10 μM DOX for 24 h before analysis
Moreover, in HT29 and LOVO cells after 72 h of incubation with 25 and 50 μM 5-FU, the extracellular levels of 5-FU were significantly higher in the supernatants of both HT29 and LOVO cells cultured in HG condition: in HT29 cells, for 25 μM dose: 21.9 ± 1.07 in G vs. 25.9 ± 2.6 in HG and for 50 μM dose: 36.1 ± 1.3 in G vs. 48.1 ± 1.2) (
Effect of normal glucose (G) and high glucose (HG) on accumulation of 5-FU in human colon cancer HT29 and LOVO cells. Cells were cultured for ≥7 days in the presence of G and HG and incubated with 25 and 50 μM 5-FU for 72 h before analysis, then subjected to the following investigations.
To confirm that the protective effect of hyperglycemia is independent of the intracellular amount of the chemotherapeutics, we observed that in HT29 cells the amount of intracellular of rhodamine 123, taken as an index of Pgp plus MRP activity, and Hoechst 33342, taken as an index of BCRP activity (
ATP binding cassette transporters activity
These data showed that very HG alone, in our experimental conditions, changed neither the intracellular accumulation of DOX nor the expression of DOX and 5-FU-effluxing membrane pumps in HT29 cells, but it decreased the amount of intracellular DOX and 5-FU by increasing the activity of MRP1 through the up regulation of MRP1 expression in LOVO cells.
According to these results, HG seemed to promote drug chemoresistance in both HT29 and LOVO cells, but in a different way. If the increased expression and activity of MRP1 in LOVO cells cultured in HG could partially explain the decreased intracellular amount of DOX, the effectiveness of DOX on HT29 cells cultured in HG is to be attributed to other molecular mechanisms, among them changes in expression/function of pro- and anti-apoptotic factors, defects in the cell cycle, and/or in expression/function of the molecular targets of anticancer drugs, and enhanced ability of cancer cells to repair anticancer drug-induced DNA damage (
To verify the effect of hyperglycemia on ROS-mediated mitochondrial pathway, we focused our attention on the expression of pro- (Bax) and anti-apoptotic (Bcl-2 and Bcl-XL) proteins that are involved not only in permeability of the outer mitochondrial membrane, but also, through their effectors, in the regulation of the cell cycle, DNA repair and replication (
Effect of normal glucose (G) and high glucose (HG) on Bcl-2, Bcl-XL, Bax, PARP and cyt c protein expression in absence or presence of DOX in human colon cancer HT29 cells. Cells were cultured for ≥7 days in the presence of G and HG, incubated for 24 h before analysis with 5 and 10 μM DOX, then washed and lysed. The level of GAPDH, used as an housekeeping protein in total lysates, and the level of β-tubulin, used as an housekeeping protein in mitochondrial lysates, were used to check the equal protein loading. The figure is representative of three independent experiments.
Densitometry of Bcl-2, Bcl-XL, Bax, PARP, and cyt c protein expression in absence or presence of DOX in human colon cancer HT29 cells in G and HG conditions. The protein bands of three independent experiments have been quantified by densitometry and the values are expressed as arbitrary units.
Effect of normal glucose (G) and high glucose (HG) on Bcl-2, Bcl-XL, Bax, PARP and cyt c protein expression in absence or presence of DOX in human colon cancer LOVO cells. Cells were cultured for ≥7 days in the presence of G and HG, incubated for 24 h before analysis with 5 and 10 μM DOX, then washed and lysed. The level of GAPDH, used as an housekeeping protein in total lysates, and the level of β-tubulin, used as an housekeeping protein in mitochondrial lysates, were used to check the equal protein loading. The figure is representative of three independent experiments.
Densitometry of Bcl-2, Bcl-XL, Bax, PARP and cyt c protein expression in absence or presence of DOX in human colon cancer LOVO cells in G and HG conditions. The protein bands of three independent experiments have been quantified by densitometry and the values are expressed as arbitrary units.
Furthermore, there was a significant decrease in the release of cytochrome c (cyt c) in the cytosol after bolus of DOX 5 and 10 μM and 5-FU 25 and 50 μM in HT29 and LOVO cells cultured in HG. This data is in agreement with the decreased expression of Bax, known mediator of the release of some factors, including the cyt c, able to trigger apoptosis if they are located in the cytosol (
We next used Poly ADP-ribose polymerase (PARP) as caspase activation index and as enzyme involved in DNA repair. In hyperglycemia, we did not observe any enzymatic cleavage of full length of PARP after DOX and 5-FU incubation of HT29 and LOVO cells, whereas in euglycemia the presence of DOX for 24 h and 5-FU for 72 h lead to a decrease (5 μM DOX, 25 μM 5-FU) or disappearance (10 μM DOX, 50 μM 5-FU) of the full length PARP in HT29 cells (
As these anti-apoptotic factors are also involved in the regulation of the cell cycle, we then performed experiments to evaluate cell cycle in euglycemic and hyperglycemic conditions.
In HT29 and LOVO cells cultured in normal glucose the 24 h DOX treatment at 10 μM dose induced a significant accumulation of the cells in the G2/M phase, demonstrating inhibition of cell proliferation by cycle arrest in this phase, accompanied by a corresponding decrease of G0/G1 population, and causing cell death as shown by an increase in the percentage of cells in sub-G1 peak (
Effect of normal glucose (G) and high glucose (HG) on cell cycle in HT29 and LOVO cells in absence or presence of DOX
In HT29 and LOVO cells cultured in normal glucose a 72 h treatment with 50 μM 5-FU led to a significant increase in the percentage of cells in S-phase suggesting a strong cell cycle arrest in this phase and a corresponding significant decrease of G0/G1 population, accompanied by a significant increase of sub-G1 cell death population (
As DOX could act as an intercalating agent of DNA synthesis blocking its transcription and inhibiting the nuclear enzyme topoisomerase activity of type II (Topo II alpha), we evaluated the effect of hyperglycemia on the Topo II alpha expression and activity. The RT-PCR results showed that hyperglycemia decreased the expression of TOPO II alpha mRNA both in HT29 and LOVO cells (
Effect of normal glucose (G) and high glucose (HG) on mRNA expression and in absence or presence of DOX in human colon cancer (HT29
In HT29 and LOVO cells (
Effect of normal glucose (G) and high glucose (HG) on Topoisomerase II alpha activity in absence or presence of DOX in human colon cancer (HT29
Many epidemiological data clearly indicate that the risk of several types of cancer is increased in diabetic patients and a number of cancer types have shown a higher mortality rate in patients with hyperglycemic associated pathologies (
Nowadays, some studies show that hyperglycemia confers resistance to drug-induced cell death in different cancer cells through different mechanisms.
ROS may play dual role in cancer progression in a dose-dependent manner. On the one hand, mild intracellular ROS can stimulate tumor progression by promoting cell proliferation, survival, invasion and metastasis; on the other hand, excess ROS production can cause oxidative damage and trigger cancer cell death (
For the first time, we demonstrate that a prolonged HG exposure protects human colon adenocarcinoma HT29 and LOVO cells against the decreased cell viability and cytotoxicity by, at least partly, lowering mitochondrial ROS production induced by DOX and 5-FU, thus impairing the effectiveness of the chemotherapy itself. Moreover, our results provide support for the role of mitochondrial derived ROS drug-induced in mtDNA damage during G growth condition, whereas under HG exposure, the small amount of generated ROS within the mitochondria leads to a low level of mtDNA damage in the D-loop and ATPase6 regions.
In addition to this evidence, as we could not exclude that HG exposure also acted on the onset of MDR through the overexpression of ABC transporters, we investigated whether the effect of hyperglycemia on the decreased cell viability and cytotoxicity induced by DOX and 5-FU may be explained by a change in the expression and activity of Pgp and/or the MDR-related proteins, among which MRP1, 5 and 8, and BCRP. We do not observe any difference neither in the amount of intracellular content of DOX and their metabolites nor in the localization of DOX when HT29 cells are cultured in G or HG conditions. In agreement with these results, we observe that in HT29 cells the amount of intracellular of rhodamine 123, taken as an index of Pgp plus MRP activity, and Hoechst 33342, taken as an index of BCRP activity, and MRP1, 5 and 8, BCRP and Pgp gene expression are not modified by HG incubation in HT29 cells. However, in LOVO cells, we observe that HG exposure decreases the amount of intracellular DOX, increasing the activity of MRP1 through the up regulation of MRP1 expression. These latter data with the results obtained in a murine tumor model in which glucose-exposed tumor cells of late tumor-bearing stage show a declined susceptibility to the cytotoxic action of cisplatin and methotrexate, accompanied by an increased expression of MDR-1 gene (
At this stage, we have no explanation on why HG induces drug chemo-resistance in a different manner in HT29 and LOVO cells. Nevertheless, if the effect of HG in LOVO cells on the response to chemotherapeutic drugs seems to follow a well-honed mechanism, the effectiveness of DOX and 5-FU on HT29 cells cultured in HG is to be attributed to other molecular mechanisms, independent from the overexpression ABC transporters, among them changes in expression/function of pro- and anti-apoptotic factors, defects in the cell cycle, and/or in expression/function of the molecular targets of anticancer drugs and enhanced ability of cancer cells to repair anticancer drug-induced DNA damage (
As is the case with most of the chemotherapeutic agents, multiple mechanisms have been implicated in the development of drugs resistance, including elevated levels of anti-apoptotic genes, alterations in permeability of the outer mitochondrial membrane and through their effectors in the regulation of the cell cycle (
The intracellular effects of DOX include free radical formation, activation of caspases, cleavage of Poly ADP-ribose polymerase (PARP), inhibition of DNA topoisomerase II and also nucleotide intercalation, resulting in inhibition of DNA replication, presence of DNA fragmentation and sub-diploid DNA content (
We therefore focus our attention on the expression of pro- (Bax) and anti-apoptotic (Bcl-2 and Bcl-XL) proteins and, as expected, we confirm that the state of hyperglycemia abolishes the pro-apoptotic effects of DOX and 5-FU by decreasing, in both HT29 and LOVO cells, the ratio Bax/Bcl-2 and Bax/Bcl-XL, taken as an indicator of susceptibility to apoptosis. In agreement with this, the release of cytochrome c in the cytosol after bolus of DOX and 5-FU is decreased in HG condition. Moreover, the basal level PARP, used as caspase activation index and as enzyme involved in DNA repair, is significantly higher in hyperglycemia and we did not observed in this condition any enzymatic cleavage of full length of PARP after DOX and 5-FU incubation of HT29 and LOVO cells.
Three of the 5-FU intracellular formed nucleotides metabolites, the 5-fluorouridine 5-triphosphate (FUTP), the 5-fluoro-2-deoxyuridine 5-triphosphate (FdUTP), and the 5-fluoro-2-deoxyuridine 5-monophosphate (FdUMP), are responsible for the antineoplastic effect of 5-FU. In brief, FUTP is incorporated into RNA and interferes with normal RNA processing and function, FdUTP is incorporated into DNA, leading to pathological DNA structures and ultimately cell death and FdUMP acts during the S phase of the cell cycle inhibiting DNA synthesis by restricting availability of thymidylate as it inhibits the thymidylate synthetase (
Taken together, these data bring further knowledge on the molecular mechanisms involved in chemoresistance during hyperglycemia. Indeed a prolonged exposure to HG protects human colon adenocarcinoma cells from the cytotoxic effects of two widely used chemotherapeutic drugs, impairing the effectiveness of the chemotherapy itself.
This should elicit an even greater care in maintaining the euglycemic state in pre- and diabetic oncological patients to increase the success of chemotherapy, thus limiting the over dose of the treatment with the subsequent side effects.
LB performed the following experiments: measurement of total cellular and mitochondrial ROS production, mitochondrial DNA (mtDNA) damage, topoisomerase II alpha assay, real-time polymerase chain reaction, and western blot experiments. She contributed to the statistical analysis, the interpretation of the results, and to write the manuscript. EM performed the following experiments: cell viability, lactate dehydrogenase (LDH) leakage and DOX accumulation and Pgp, MRP and BCRP activities and western blot experiments. She participated in the discussion of the results and contributed to draft the manuscript. RM performed the following experiments: cell viability, lactate dehydrogenase (LDH) leakage and DOX accumulation. OB performed preparation of samples and the measurements of cell cycle. She discussed the results concerning cell cycle analysis. BR performed preparation of samples and the measurements of RP-HPLC quantification of DOX and 5-FU and their metabolites. She discussed the results concerning RP-HPLC analysis. SD conceived the study, performed immunofluorescence staining experiments, contributed to the final interpretation of the data and to write the manuscript. All authors have read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
SD was supported by public university funds of University of Turin. LB was funded by the post-doc program of the University of Turin. EM was funded by the Ph.D. program of the University of Turin.
The Supplementary Material for this article can be found online at:
Effect of normal glucose (G) and high glucose (HG) on Bcl-2, Bcl-XL, Bax, PARP, and cyt c protein expression in absence or presence of 5-FU in human colon cancer HT29 cells. Cells were cultured for ≥7 days in the presence of G and HG, incubated for 24 h before analysis with 25 and 50 μM 5-FU, then washed and lysed. The level of GAPDH, used as an housekeeping protein in total lysates, and the level of β-tubulin, used as an housekeeping protein in mitochondrial lysates, were used to check the equal protein loading. The figure is representative of two independent experiments.
Densitometry of Bcl-2, Bcl-XL, Bax, PARP, and cyt c protein expression in absence or presence of 5-FU in human colon cancer HT29 cells in G and HG conditions. The protein bands of two independent experiments have been quantified by densitometry and the values are expressed as arbitrary units.
Effect of normal glucose (G) and high glucose (HG) on Bcl-2, Bcl-XL, Bax, PARP, and cyt c protein expression in absence or presence of 5-FU (5-FU) in human colon cancer LOVO cells. Cells were cultured for ≥7 days in the presence of G and HG, incubated for 24 h before analysis with 25 and 50 μM 5-FU, then washed and lysed. The level of GAPDH, used as an housekeeping protein in total lysates, and the level of β-tubulin, used as an housekeeping protein in mitochondrial lysates, were used to check the equal protein loading. The figure is representative of three independent experiments.
Densitometry of Bcl-2, Bcl-XL, Bax, PARP, and cyt c protein expression in absence or presence of 5-FU in human colon cancer LOVO cells in G and HG conditions. The protein bands of two independent experiments have been quantified by densitometry and the values are expressed as arbitrary units.