Various cancer cells require massive amounts of glucose as an energy source for their dysregulated growth. Although D-allose, a rare sugar, inhibits tumor cell growth via inhibition of glucose uptake, a few cells can survive after treatment. However, the mechanism by which D-allose-resistant cells are generated remains unclear. Here, we investigated the properties of D-allose-resistant cells and evaluated the efficacy of combined treatment with this rare sugar and antitumor drugs. To this end, we established a D-allose-resistant tumor cell line and prepared a C57BL/6J mouse tumor xenograft model using Lewis lung carcinoma (LLC) cells. Xenograft-bearing mice were treated with D-allose (9 g/kg) and/or hydroxychloroquine (HCQ, 60 mg/kg), an autophagy inhibitor, for two weeks. Although D-allose inhibited LLC cell growth in a dose-dependent manner, a few cells survived. The upregulation of LC3-II, a classical autophagy marker, and the downregulation of mTOR and its downstream molecule Beclin1 were observed in established D-allose-resistant LLC cells, which were more sensitive to cell death induced by HCQ. Similarly, in the tumor xenograft model, the tumor volume in mice co-treated with D-allose and HCQ was considerably smaller than that in untreated or HCQ-treated mice. Importantly, the administration of D-allose induced autophagy selectively at the tumor site of the xenograft-bearing mice. These results provide a new therapeutic strategy targeting autophagy which is induced in tumor cells by D-allose administration, and may be used to improve therapies for lung cancer.
Lung cancer is associated with high morbidity and mortality worldwide, with 1.8 million estimated deaths due to lung cancer as a primary condition in 2020 (
Recently, the use of rare sugars that are defined as monosaccharides and their rare derivatives has attracted attention for their various physiological functions. Among these sugars, D-allose has a sweetness of 80% compared to that of sugar (
Tumor cells have acquired numerous functions to enable their survival under hypoxic and hypotrophic conditions in the microenvironment. For example, under hypoxic conditions, tumor cells adapt to the microenvironment by increasing the expression of hypoxia-inducible factor-1 (HIF-1) (
In the present study, we found that although D-allose killed most of the tumor cells, a few cells induced autophagy to survive. Furthermore, we showed that a combined treatment with D-allose and the autophagy inhibitor hydroxychloroquine (HCQ) significantly suppressed tumor cell growth without any side effects in a mouse tumor model.
The monosaccharides used in this study are listed in
Mouse Lewis lung carcinoma (LLC) cells were purchased from Riken BioResource Center (catalog no. RCB0558, RRID: CVCL_4358) and mouse skin melanoma (B16F10) cells were obtained from the American Type Culture Collection (ATCC) (catalog no. CRL-6475). The MDA-MB-231 human breast adenocarcinoma cell line was purchased from the Japanese Cancer Research Resources Bank (catalog no. JCRB1559). LLC, B16F10, and MDA-MB-231 cells were maintained in RPMI-1640 or low-glucose DMEM (Fujifilm Wako Pure Chemical, Ltd.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and penicillin (100 units/ml)-streptomycin (0.1 mg/ml) (Life Technologies/Thermo Fisher Scientific, Inc.).
The cells were given fresh culture media twice per week and were subcultured to confluency after detaching the cells with 0.25% trypsin +0.02% EDTA at a weekly split ratio of ~1:2. Cultures from passages 10 to 25 were used in all experiments. The cells were screened periodically for mycoplasma contamination using a Mycoplasma Detection kit (MycoAlert™; Lonza Group, Ltd.).
For the cell viability assay, cells (1×105/well) were seeded in 6-well plates and cultured for 24 h at 37°C in 5% CO2. Monosaccharides dissolved in PBS were added to form a final concentration of 25 or 50 mM. Stocks of monosaccharides (500 mM) were prepared in RPMI-1640 medium and sterilized via filtration through a 0.2-µm pore filter. For the control conditions, the same volume of PBS was added. The viable cells were enumerated via 0.5% trypan blue staining (Nacalai Tesque).
D-allose-resistant LLC cells were established using the following procedure: Untreated LLC cells were seeded at a density of 2×104 cells/ml in a 100-mm dish. D-allose (25 mM) dissolved in the RPMI-1640 medium was added. After 72 h, cells were harvested and enumerated via 0.5% trypan blue staining, adjusted to a density of 2×104 cells/ml, and reseeded in the presence of 25 mM D-allose. The first cell count was denoted to be passage 1, and after counts until passage 10 when the cell ratio of the control LLC cells and D-allose-resistant LLC cells exceeded 100%, the population was considered to comprise D-allose-resistant cells. Furthermore, we confirmed that D-allose-resistant LLC cells were stably resistant at least until passage 10 after establishment.
The collected cells and tumor tissues were homogenized for 1 min at 4°C using an ultrasonic homogenizer in 9 volumes of 50 mM Tris-HCl (pH 6.8) containing 1% sodium dihydrogen phosphate, 1% protease inhibitor cocktail (Nacalai Tesque), and 1% EDTA-free phosphatase inhibitor cocktail (Nacalai Tesque). The homogenized samples were centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was collected as a sample extract. The protein concentration in the sample extract was measured using a bicinchoninic acid protein assay kit (Takara). The protein for loading was prepared by adding bromophenol blue/2-mercaptoethanol corresponding to 0.1 volumes of the final sample volume. The amount of protein loaded per lane was 0.5–40 µg (0.5 µg for Akt, p-Akt, and Beclin1, 5 µg for LC3 and p62, 40 µg for mTOR and p-mTOR). The protein samples were separated via 7.5–15% gels (7.5% for p62, mTOR, and p-mTOR, 10% for Akt, p-Akt, and Beclin1, 15% for LC3). Proteins were separated via SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk (for LC3, p62, Akt, Beclin1, and β-actin) or 5% PhosphoBLOCKER Blocking Reagent (for mTOR, p-Akt, and p-mTOR; Cell Biolabs Inc.), diluted in TBS-T for 1 h at 25°C, and then incubated with a primary antibody. The following antibodies were used at a dilution of 1:1,000: anti-LC3A/B (catalog no. #12741; Cell Signaling Technology, Inc.), anti-SQSTM1/p62 (catalog no. #5114; Cell Signaling Technology, Inc.), anti-mTOR (catalog no. #2972; Cell Signaling Technology, Inc.), anti-phospho-mTOR (catalog no. #5536; Cell Signaling Technology, Inc.), anti-Beclin1 (catalog no. #3738; Cell Signaling Technology, Inc.), anti-Akt (catalog no. #4691; Cell Signaling Technology, Inc.), anti-phospho-Akt (catalog no. #4060; Cell Signaling Technology, Inc.), and anti-β-actin (catalog no. A5441; Sigma-Aldrich/Merck KGaA) and were incubated overnight at 4°C. The membranes were subsequently washed and incubated for 1 h with a secondary HRP-conjugated antibody (1:10,000; catalog no. 115-035-144; Jackson ImmunoResearch Inc.). Immunolabeling was performed using an enhanced chemiluminescence detection system (GE Healthcare). The band intensities of the detected proteins (or the phosphorylated proteins) were analyzed via densitometry using the ImageJ software v1.53q (National Institutes of Health) and normalized to those of β-actin. To re-probe the PVDF membranes, the antibodies bound to the membranes were removed by washing twice (15 min each) with a commercial stripping solution and twice (15 min each) with TBS-T, and then the blotted membranes were re-blocked with BSA and re-probed with anti-β-actin antibody.
Cells were fixed with 100% methanol for 20 min and then permeabilized using 0.1% Triton X-100 for an additional 30 min. After incubation in blocking solution (1% BSA in 0.1% Tween 20/PBS) for 1 h, anti-LC3 A/B (1:100; catalog no. #12741; Cell Signaling Technology) and anti-β-actin (1:1,000; catalog no. #A5441; Sigma-Aldrich/Merck KGaA) antibodies diluted in blocking solution were applied and incubated for 1 h at 25°C. After washing in PBS, the cells were incubated with secondary antibodies (1:1,000; Alexa 568- conjugated anti-rabbit IgG, catalog no. #A31628; and Alexa 488-conjugated anti-mouse IgG, catalog no. A10037; Invitrogen/Thermo Fisher Scientific, Inc.) for 1 h at 25°C. Nuclei were stained with DAPI (1:1,000; Dojindo Laboratories, Inc.) for 5 min. Immunofluorescence signals were observed using a Keyence BZ-9000 fluorescence microscope (magnification, ×600; Keyence Corp.).
Cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 16 h at 4°C, and then rinsed three times with 0.1 M PB. Post-fixation was performed with 1% osmium tetroxide/PB for 1 h on ice, and the cells were dehydrated in graded ethanol on ice and embedded in EPON 812 epoxy resin (TAAB) at 60°C for 72 h. Ultrathin sections (80–100 nm) were cut with an ultramicrotome (EM UC7, Leica Microsystems GmbH) and collected on copper grids. The ultrathin sections were double-stained with uranyl acetate and lead citrate and were subsequently observed on a transmission electron microscope (JEM1400-Flash, JEOL, Ltd.).
Total RNA was extracted from LLC cells using the FastGene™ RNA Basic Kit (Nippon Genetics Co. Ltd.), and the RNA concentration was determined spectrophotometrically (NanoDrop One, Thermo Fisher Scientific, Inc.) at 260 nm. The RNA (500 ng) was then used for first-strand synthesis of cDNA using ReverTra Ace qPCR RT Master Mix (Toyobo Life Science). The following PCR primers were used in this study:
Seven-week-old male C57BL/6J mice (weighing 18.0-22.0 g) were purchased from Charles River Laboratories (Yokohama, Japan). Fifty mice were used in this experiment. Mice were housed in plastic cages (five mice/cage) under controlled conditions of light (12-h light/dark cycle), temperature (23±2°C), and humidity (55%), and had free access to food and water. The protocols for all animal experiments were approved by the Animal Experimentation Committee of Fujita Health University (approval no. AP19053). Procedures involving mice and their care conformed to international guidelines, as described in the Principles of Laboratory Animal Care (National Institutes of Health publication 85–23, revised 1985 (
C57BL/6J mice were acclimated for one week in a rearing environment. Logarithmic growth phase LLC cells (5×105 cells/100 µl) were subcutaneously injected into the right posterior flank of each mouse. When the tumor volume reached approximately 50 mm3, the mice were randomly assigned to four groups. D-allose was orally administered daily at 9 g/kg (D-allose group, n=10) for two weeks as previously described (
Serum D-allose content in samples was analyzed via HPLC. Briefly, serum samples were mixed (1:1) in 0.6 M perchloric acid. The resulting supernatant (20 µl) was subjected to HPLC analysis. Jusco Finepak GEL SA-121 column (6×100 mm) maintained at 80°C was used as the anion exchange column. Elution was performed using a gradient of solvent A (0.25 M sodium borate buffer; pH 7.5), and solvent B (0.6 M sodium borate buffer; pH 7.5) at a flow rate of 0.40 ml/min. The gradient changed from 70% A/30% B to 50% A/50% B for 20 min, 50% A/50% B to 0% A/100% B for 1 min; the gradient was maintained at 0% A/100% B for 17 min; and then the gradient changed from 0% A/100% B to 70% A/30% B for 2 min. The eluate from the column was admixed with guanidine-acetonitrile (pH 11.0) at a flow rate of 0.60 ml/min. The resultant effluent was passed through a reaction coil maintained at 160°C and fluorescence was recorded using excitation and emission wavelengths of 310 and 415 nm, respectively.
Tumor tissues were fixed in 10% formalin in PBS overnight. The specimens were embedded in paraffin. Sections that were 4-µm thick were used for hematoxylin and eosin (H&E) staining and immunofluorescence analysis. For immunofluorescence staining sections were incubated in 0.1 M citrate buffer (pH 6.0) for 15 min and heated up to 121°C using an autoclave. After washing with PBS, sections were incubated in 0.3% hydrogen peroxide and methanol for 30 min to inactivate the endogenous peroxidase. Nonspecific antibody-binding sites were blocked in 2.5% normal horse serum for 30 min. The sections were subsequently incubated with rabbit anti-LC3 A/B antibody (1:1,000; catalog no. #12741; Cell Signaling Technology, Inc.) in PBS and incubated for 1 h at 25°C. After primary antibody incubation, the sections were rinsed with PBS and incubated with secondary antibody solution (ImmPRESS Reagent, Vector Laboratories, Inc.) for 30 min at 25°C, followed by the addition of 3,3′-diaminobenzidine tetrahydrochloride (Dako/Agilent Technologies, Inc.). The sections were counterstained with hematoxylin.
Mouse serum glucose (GLU), alanine aminotransferase (ALT), triglyceride (TG), total protein (TP), creatinine (CRE), and blood urea nitrogen (BUN) levels were measured using an automated chemistry analyzer (BioMajesty JCA-BM9130; Jeol Ltd.).
All results are expressed as mean ± standard error of the mean (SEM). Significant differences between three or four groups were determined using one-way ANOVA or two-way ANOVA, followed by Tukey's multiple comparison test, and those between two groups were determined using the Student's t-test. A value of P< 0.05 was considered significant. All statistical analyses were performed using GraphPad Prism v6.07 (GraphPad Software, Inc.).
To evaluate the effect of rare sugars on tumor growth, we cultured LLC cells in the presence of various rare sugars with D-glucose as a control. D-mannose, D-allose, L-psicose, D-arabinose, L-arabinose, and L-fucose suppressed cell growth compared to growth in the presence of PBS or D-glucose (
D-allose treatment inhibited LLC cell growth in a significantly dose-dependent manner, and the effect persisted for at least 72 h after treatment (
To investigate whether D-allose-induced autophagy is essential for tumor cell survival, we established D-allose-resistant LLC cell lines by selecting only LLC cells that survived in the long-term D-allose cultures. Compared to the untreated control cells, when the proportion of the D-allose-treated LLC cells exceeded 100%, the cells were determined to be D-allose-resistant LLC cells (
HCQ, an autophagy inhibitor, is currently studied in phase I and II clinical trials, and more than 20 trials involving HCQ have recruited patients with cancer worldwide. Several studies have shown evidence of preliminary antitumor activity (
Several studies indicate that the mTOR-dependent pathway is a key regulator of autophagy (
To determine whether a combination therapy of D-allose and HCQ shows enhanced antitumor activity
In the present study, we found that D-allose inhibited the growth of various tumor cells including mouse- and human-derived tumor cells and induced autophagy in the surviving cells. Furthermore, the enhanced autophagy in the established D-allose-resistant tumor cells was associated with increased sensitivity to hydroxychloroquine (HCQ), leading to the induction of autophagic cell death. These results indicate that the combination of D-allose and HCQ can significantly inhibit tumor growth in a mouse tumor-bearing model without causing significant side effects.
Autophagy is induced by several pathways via mTOR signaling, including the PI3K/AKT (
We showed that the D-allose-induced downregulation of mTOR protein is positively correlated with the upregulation of LC-II and Beclin1 as well as the increased number of autophagosomes in the resistant Lewis lung carcinoma (LLC) cells, and the sensitivity to HCQ in the resistant LLC cells was enhanced, indicating that D-allose-induced autophagy not only is essential for their survival but is also a promising therapeutic target. Our findings raise a question regarding which cells acquire resistance against D-allose via the induction of autophagy. Based on this aspect, it is known that autophagy is induced constitutively and predominantly in cancer stem cells to maintain their survival and pluripotency (
We also found that LLC cell proliferation was considerably inhibited in the D-allose and HCQ co-treatment group compared to that in the control and HCQ groups. Based on our findings, we propose that D-allose kills non-stem cancer cells and sensitizes surviving cancer stem cells to HCQ chemotherapy, leading to the suppression of tumor growth. Candidate autophagy inhibitors for cancer treatment include bafilomycin A1, 3-methyladenine, chloroquine (CQ), and HCQ, among which HCQ is currently undergoing phase II clinical trials (
This study provides a new therapeutic strategy that targets autophagy induced in tumor cells by D-allose administration. Notably, D-allose, which has various physiological functions, can be mass-produced industrially; however, there are no studies focusing on autophagy in tumor cells. Therefore, the new information on D-allose found in this study will contribute to improving the therapeutic response in combination with clinically applied autophagy inhibitors.
Not appliable.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
MHo and KS planned the experiments. KY, MHo, HT, NM, MHi, SY, and FS performed the experiments. KY, MHo, and HT were responsible for data integrity and data analysis. KY, MHo, HT, NM, MHi, FS, SY, and KS discussed the results. KY, MHo, and HT wrote the manuscript. KY, MHo, HT, NM, MHi, FS, SY, and KS conducted the research. KY and MHo confirm the authenticity of all the raw data. KS had primary responsibility for the final content. All authors read and approved the final manuscript for publication.
The protocols for all animal experiments were approved by the Animal Experimentation Committee of Fujita Health University (approval no. AP19053).
Not applicable.
Kyoka Yamazaki is an employee of Matsutani Chemical Industry Co., Ltd. Kuniaki Saito was funded by A&T Corporation, Nisshin Seifun Group, Marukome Corporation, and Tsuji-seiyu Corporation and belongs to an endowed chair funded by Fujifilm Wako Pure Chemical Corporation and A&T Corporation. The other authors have no financial competing interests.
D-allose impairs cell growth and promotes autophagy in LLC cells. LLC cells were cultured for 48 h with or without 25 mM of several monosaccharides. (A and B) The number of live LLC cells was counted using trypan blue staining. (C) Growth curves of LLC cells treated with or without 50 mM D-glucose or 50 mM D-allose. (D) Protein extracts from LLC cells were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and immunoblotting was performed using anti-LC3 and anti-p62 antibodies. The results are presented as representative data. The relative densitometric intensities of LC3-II and p62 were determined for each protein band and normalized to that of β-actin. Data are presented as the mean ± SEM. Statistical analysis was performed using (B and D) one-way ANOVA with Tukey's multiple comparison tests or (C) two-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01. LLC, Lewis lung carcinoma cells.
LLC cells surviving under continuous treatment of D-allose promote autophagy and show enhanced susceptibility to autophagy inhibitor hydroxychloroquine. (A) D-allose-resistant LLC cells were established via selection of LLC cells surviving under continuous treatment with 25 mM D-allose. (B) Protein extracts from D-allose-resistant LLC cells were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and immunoblotting was performed using anti-LC3 antibodies. The results are presented as representative data (left). The relative densitometric intensity of LC3-II was determined for each protein band and normalized to β-actin (right). (C) LLC cells (control) and D-allose-resistant LLC cells were stained for LC3, β-actin, and 4′,6-diamidino-2-phenylindole (nuclei) using immunofluorescence staining. (D) Representative images of transmission electron microscopy of autophagic ultrastructural features in LLC cells (control) and D-allose-resistant LLC cells. Red arrows indicate autolysosomes and autophagosomes. Scale bars, 2.5 µm (left) or 1.0 µm (right). (E) The efficacy of hydroxychloroquine (5 nM) in D-allose-resistant LLC cells was determined by enumerating the live cells using trypan blue staining. Data are presented as the mean ± SEM (n=3). Statistical analysis was performed using paired two-tailed Student's t-test. *P<0.05, **P<0.01 vs. the control group. LLC, Lewis lung carcinoma cells.
D-allose-resistant LLC cells contain reduced mTOR levels. (A) Protein extracts from LLC cells (control) and D-allose-resistant LLC cells were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and immunoblotting was performed using anti-Akt, -p-Akt, -mTOR, -p-mTOR, -Beclin1 and -β-actin antibodies. The results are shown as representative data. (B) The relative densitometric intensity of Akt, p-Akt, mTOR, p-mTOR, and Beclin1 was determined for each protein band and normalized to that of β-actin. (C) The levels of
Combination therapy of D-allose and hydroxychloroquine (HCQ) reduces LLC cell growth in mice. Changes in (A) body weight and (B) tumor growth curves in LLC xenograft mice following initiation of treatment without (control, n=9) or with D-allose (9 g/kg, n=10), HCQ (60 mg/kg, n=10), and a combination of D-allose (9 g/kg) and HCQ (60 mg/kg) (n=10). Data are presented as the mean ± SEM. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. *P<0.05, **P<0.01 vs. the control, #P<0.05, ##P<0.01 vs. HCQ treatment. (C) Representative images of frozen tumor tissues (cut in the half size) of indicated mice 14 days after treatment are shown. (D) Serum D-allose levels in control and D-allose-treated mice 14 days after treatment were measured by high-performance liquid chromatography. **P<0.01 vs. the control. (E and F) Expression levels of LC3-II in the tumor tissues of control and D-allose-treated mice 14 days after treatment. The results are presented as representative data (left). The relative densitometric intensity of LC3-II was determined for each protein band and normalized to that of β-actin (right). Data are presented as the mean ± SEM. Statistical analysis was performed using a paired two-tailed Student's t-test. *P<0.05 vs. the control. (G) LC-3 in tumor sites of control and D-allose-treated mice 14 days after treatment was detected using immunohistochemistry staining. Hematoxylin and eosin (H&E) staining results are shown in the right column. HCQ, hydroxychloroquine; LLC, Lewis lung carcinoma.
Monosaccharides used in this study.
Monosaccharides | Category | Molecular weight |
---|---|---|
D-glucose | Aldohexose | 180.16 |
D-mannose | Aldohexose | 180.16 |
D-allose | Aldohexose | 180.16 |
D-psicose | Ketohexose | 180.16 |
D-tagatose | Ketohexose | 180.16 |
D-sorbose | Ketohexose | 180.16 |
L-psicose | Ketohexose | 180.16 |
D-arabinose | Aldopentose | 150.13 |
L-arabinose | Aldopentose | 150.13 |
L-fucose | Deoxy sugar | 164.16 |
Nutritional status, liver function, and renal function in mice after 14 days of treatment.
Control (No treatment) n=9 | D-allose n=10 | HCQ n=10 | D-allose + HCQ n=10 | |
---|---|---|---|---|
GLU (mg/dl) | 170.0±7.82 | 174.5±12.44 | 181.5±11.94 | 193.5±6.71 |
ALT (U/l) | 10.0±1.57 | 14.0±2.43 | 15.5±1.80 | 17.5±3.69 |
TG (mg/dl) | 183.9±53.57 | 212.5±32.33 | 131.5±31.50 | 217.5±56.90 |
TP (g/dl) | 5.11±0.19 | 5.30±0.14 | 5.65±0.17 | 5.53±0.05 |
CRE (mg/dl) | 0.14±0.01 | 0.15±0.01 | 0.15±0.00 | 0.14±0.01 |
BUN (mg/dl) | 33.06±1.93 | 32.05±2.14 | 32.25±1.79 | 30.05±1.27 |
Data are presented as the mean ± SEM (n=9-10) and analyzed via one-way ANOVA with Tukey's multiple comparison test. HCQ, hydroxychloroquine; GLU, glucose; ALT, alanine aminotransferase; TG, triglyceride; TP, total protein; CRE, creatinine; BUN, blood urea nitrogen.