Research ArticleExperimental Studies

8-Chloroadenosine Induces ER Stress and Apoptotic Cell Death in Cholangiocarcinoma Cells

JOMNARONG LERTSUWAN, JISNUSON SVASTI and JUTAMAAD SATAYAVIVAD
Anticancer Research December 2023, 43 (12) 5425-5436; DOI: https://doi.org/10.21873/anticanres.16746

Abstract

Background/Aim: Cholangiocarcinoma is a lethal cancer, and current chemotherapeutic drugs are not very effective. Recent studies reported that cholangiocarcinoma cells were sensitive to adenosine. One adenosine analog, 8-chloroadenosine (8-CA), was shown to be more potent than adenosine and induced apoptosis in leukemia cells. This study examined effects of 8-CA in cholangiocarcinoma cells and immortalized cholangiocytes. Materials and Methods: Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell invasion was examined by transwell assay. Cell cycle and cell death were evaluated by flow cytometry. Colorimetric absorbance assay was used to assessed RNA and protein synthesis as well as mitochondrial membrane potential. Protein levels were examined by western blot analysis. Animal experiment was performed in Balb/cAJcl-Nu mice. Results: 8-CA reduced cholangiocarcinoma cell growth, prevented colony formation and caused endoplasmic reticulum stress and cell-cycle arrest. Eventually, apoptosis was induced. However, treatment with 8-CA did not interfere with RNA synthesis or protein synthesis and did not alter mitochondrial membrane potential. Combination of 8-CA with several chemotherapeutic drugs in vitro was less effective than 8-CA alone and the drugs alone, except for the combination of 8-CA with hydroxychloroquine, which had an additive effect on RMCCA-1 cells. However, further in vivo study showed that treatment with 8-CA alone inhibited tumor growth more than treatment with a combination of 8-CA with hydroxychloroquine. Conclusion: 8-Chloroadenosine inhibited CCA cells by inducing endoplasmic reticulum stress and apoptosis. In vivo study showed that 8-CA inhibited cholangiocarcinoma tumor growth better when administered alone as compared to a combination with hydroxychloroquine.

Key Words:

Cholangiocarcinoma is one of the most lethal cancers commonly found in Asia, particularly in Thailand, Korea, and China (1-4). It is the second leading cause of cancer-related death for Thai men after lung cancer (5). Although the incidence of cholangiocarcinoma is lower in other parts of the world, its incidence has increased worldwide over the past few decades (6-10). Main risk factors include inflammation of bile ducts and bile stasis, which result from chemical exposure, parasitic fluke infection and alcohol consumption (11). Surgical resection is the primary curative treatment for patients with cholangiocarcinoma (12, 13), but only 20% of patients are able to undergo surgery (12, 14). For those who have unresectable cholangiocarcinoma, current first-line chemotherapy uses gemcitabine and cisplatin (15). Unfortunately, chemotherapy still leaves many patients with poor outcomes (8), so novel therapeutic compounds are needed.

Recent studies have shown that adenosine inhibited cholangiocarcinoma cell growth and motility (16), and induced autophagy (17). Moreover, cholangiocarcinoma is more sensitive to adenosine than some other cancers, such as breast cancer (18) and prostate cancer (19). One adenosine analog, 8-chloroadenosine (8-CA), was reported to have more potential in inhibiting cancer cells by interfering with RNA synthesis and inducing apoptosis (20). 8-CA was also reported to have inhibitory effects on breast cancer (20) and acute myeloid leukemia (21) cells, and is under clinical trial for acute myeloid leukemia (NCT02509546). We aimed to evaluate the inhibitory effects of 8-CA on cholangiocarcinoma in vitro and in vivo.

Materials and Methods

Cell lines. KKU-213, KKU-055, KKU-100 and MMNK-1 cells were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). RMCCA-1 cell line was established from a peripheral cholangiocarcinoma specimen surgically obtained from a male Thai patient (22) and was obtained from the Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok, Thailand. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco), 1% minimum essential medium, non-essential amino acid (Gibco) and 1% penicillin/streptomycin (Gibco). All cells were maintained at 37°C with 5% CO2 in a humidified incubator.

Chemicals. Chemicals in this study included 8-CA (Tocris, Bristol, UK), hydroxychloroquine (Sigma-Aldrich, Saint Louis, MO, USA), bafilomycin A1 (Abcam, Waltham, MA, USA), gemcitabine (Sigma-Aldrich), paclitaxel (Sigma-Aldrich), cisplatin (D3371; Tokyo Chemical Industry, Tokyo, Japan), 5-fluorouracil (5-FU) (Sigma-Aldrich) and silmitasertib (CX-4945), an inhibitor of protein kinase (casein kinase II) (Abcam). Chemicals were prepared in different solvents in stock at high concentration. Hydroxychloroquine was dissolved in phosphate buffered saline to make 30 mM stock. Cisplatin was dissolved in normal saline to make 10 mM stock. Bafilomycin A1, gemcitabine, paclitaxel, 5-FU and CX-4945 were dissolved in dimethyl sulfoxide to make 10 mM stock.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were plated in 96-well plates to achieve 25% confluence and allowed to adhere overnight. Media were changed and treatments were added. 8-CA was added in doses from 0.1 to 100 μM in a half-log increments and cells were incubated for 1, 2 and 3 days. For drug combination experiments, gemcitabine, cisplatin, paclitaxel, 5-FU or CX-4945 were added in doses from 0.1 to 100 μM in half-log increments simultaneously with 1, 3, or 10 μM 8-CA and cells were incubated for 24 hours. In other experiments, 30 or 50 μM hydroxychloroquine were added 4 hours prior to the 24-hour treatment with 8-CA at doses from 0.1 to 100 μM in half-log increments. The control group was treated with the solvents (vehicle) used for the drugs. In drug combination experiments, the control group, received the solvent of all drugs, and group for drug 1 also received solvent of drug 2 and vice versa. After treatment, media were removed and 100 μl of 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (Sigma Aldrich) in serum-free DMEM was added and cells were incubated for 2.5 hours at 37°C. Then 50 μl of stop solution (10% sodium dodecyl sulfate in 50% dimethylformamide in distilled water) was added and mixed thoroughly before reading the absorbance at 560 nm on a plate reader. Data were normalized to those of the control group and are presented as a percentage. Three replicates were performed.

5-Bromo-2′-deoxyuridine (BrdU) assay. Cells were plated in 96-well plates to achieve 75% confluence. After cells adhered overnight, media were changed, and 8-CA was added to a final concentration of 10 μM and cells were incubated for 24 hours. The control group was treated with the solvents (vehicle) used for the drugs. Incorporation of BrdU into DNA strands was detected by using a BrdU Cell Proliferation ELISA Kit (ab126556; Abcam). Cells were prepared according to the manufacturer’s protocol and analyzed on a plate reader. Absorbance values at 450 nM were normalized to those of the control group and are presented as a percentage. Three replicates were performed.

Invasion assay. Polycarbonate membrane Transwell inserts (8 μm; Falcon, New York, NY, USA) were coated with 50 μl of a 0.1x Matrigel (BD Biosciences, San Jose, CA, USA) in serum-free DMEM. A total of 2×104 cells in serum-free DMEM was added to the top chamber of each well. The bottom well was filled with complete DMEM. 8-CA was added at a final concentration of 10 μM to both the Transwell and the bottom well. The control group was treated with the solvents (vehicle) used for the drugs. Cells were incubated for 24 hours. Cells and Matrigel remaining on the top of the membrane were removed. Cells on the underside of the membrane were fixed in methanol for 5 minutes and stained with 0.5% (w/v) crystal violet in 12% glutaraldehyde in water for 15 minutes. Following a brief wash with distilled water, cells were air dried for 1 day and counted using a Nikon Eclipse T2S phase-contrast inverted fluorescent microscope. Numbers of invading cells were normalized to those of the control group and are presented in percentage. Three replicates were performed.

Cell-cycle analysis. Cells were plated in 96-well plates to achieve 75% confluence and allowed to adhere overnight. Media were changed and 8-CA was added to a final concentration of 10 μM and cells were incubated for 24 hours. The control group was treated with the solvents (vehicle) used for the drugs. The cell-cycle distribution was analyzed using Muse™ Cell Cycle kit (Merck Millipore, Billerica, MA, USA). Cells were detached by trypsinization and prepared according to the manufacturer’s protocol and analyzed with Luminex-Guava Muse/cell analyzer. Three replicates were performed.

Cell death analysis. Cells were plated in 96-well plates to achieve 75% confluence and allowed to adhere overnight. Media were changed and 8-CA was added to a final concentration of 10 μM and cells were incubated for 24 h. The control group was treated with the solvents (vehicle) used for the drugs. Apoptotic cell death was analyzed by using Muse™ Annexin V & Dead Cell Kit (Merck Millipore). Cells were detached by trypsinization, fixed, and stained according to the manufacturer’s protocol, and analyzed with Luminex-Guava Muse/cell analyzer. Three replicates were performed.

Colony formation. Cells were plated in 24-well plates to achieve 75% confluence and allowed to adhere overnight. Media were changed and 8-CA was added to a final concentration of 10 μM and cells were incubated for 24 h. The control group was treated with the solvents (vehicle) used for the drugs. Cells were detached by trypsinization and stained with trypan blue to check cell viability. Five hundred viable cells were transferred to 6-well plates. Cells were incubated in complete DMEM for 10 days without treatment. Media were changed three times a week. After that, cells were fixed in methanol for 5 min and stained with 0.5% (w/v) crystal violet in 12% glutaraldehyde in water for 15 min. Following a brief wash with distilled water, plates were air dried for 1 day. Colonies were counted using a Nikon Eclipse T2S phase-contrast inverted fluorescence microscope. Groups of 30 cells or more were counted as a colony. Numbers of colonies were normalized to the control group and presented in percentage. Three replicates were performed.

RNA synthesis. Cells were plated in 96-well plates to achieve 75% confluence and allowed to adhere overnight. Media were changed and 8-CA was added to a final concentration of 10 μM and cells were incubated for 24 hours. The control group was treated with the solvents (vehicle) used for the drugs. RNA synthesis was measured by using RNA Synthesis Assay Kit (ab228561; Abcam). Cells were prepared according to the manufacturer’s protocol and analyzed on a plate reader. Actinomycin D was used as a positive control. Fluorescence signals (excitation/emission=480/530 nm) were normalized to those of the control group and are presented as a percentage. Three replicates were performed.

Protein synthesis. Cells were plated in 96-well plates to achieve 75% confluence and allowed to adhere overnight. Media were changed and 8-CA was added to a final concentration of 10 μM and cells were incubated for 24 hours. The control group was treated with the solvents (vehicle) used for the drugs. Protein synthesis was measured by using protein synthesis assay kit (AB239725; Abcam). Cells were prepared according to the manufacturer’s protocol and analyzed on a plate reader. Cycloheximide was used as a positive control. Fluorescence signals (excitation/emission=480/530 nm) were normalized to those of the control group and are presented as a percentage. Three replicates were performed.

Mitochondrial membrane potential assay. Cells were plated in 96-well plates to achieve 75% confluence and allowed to adhere overnight. Media were changed and 8-CA was added to a final concentration of 10 μM and cells were incubated for 4 or 24 h. The control group was treated with the solvents (vehicle) used for the drugs. Mitochondrial membrane potential was measured by using tetramethylrhodamine ethyl ester Mitochondrial Membrane Potential Assay Kit (ab113852; Abcam). Cells were prepared according to the manufacturer’s protocol and analyzed on a plate reader. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone was used as a positive control. Fluorescence signals (excitation/emission=549/575 nm) those of the control group and are presented as a percentage. Three replicates were performed.

Western blot. Thirty milligrams of total protein extracted from KKU-213 and RMCCA-1 cells treated with 10 μM 8-CA for 24 hours were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated for 18 hours on a rocking shaker at 4°C with primary antibodies. Primary antibodies were against microtubule-associated protein light chain 3B (LC3B) (1:1500; 3868; Cell Signaling Technology, Danvers, MA, USA), C/EBP homologous protein (CHOP) (1:1,500; 2895; Cell Signaling Technology) and β-actin (1:5,000; A2066; Sigma Aldrich). Then membranes were incubated for 75 minutes on a rocking shaker at 23°C with secondary antibodies. Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5,000; 7074, Cell Signaling Technology) or horse anti-mouse immunoglobulin G (1:5,000; 7076, Cell Signaling Technology). Signals were visualized by autoradiography. Band intensity was analyzed using ImageJ software version 1.8.0_172 (National Institutes of Health, Bethesda, MD, USA). Band intensity values from ImageJ software were normalized to the value from the control group and are presented as a percentage. Three replicates were performed.

Animal experiments. Animal experiments were performed at an Association for Assessment and Accreditation of Laboratory Animal Care-accredited Laboratory Animal Unit at the Chulabhorn Research Institute. The animal protocol was approved by the Institutional Animal Care and Use Committee (approval number 2022-05). Six-week-old male nude mice (strain: BALB/cAJcl-Nu with average weight of 25.64±1.29 g) were used (Nomura Siam International, Bangkok, Thailand). Three animals were assigned to each experimental group. There were five groups: Vehicle control, 15 mg/kg/week 8-CA, 45 mg/kg/week 8-CA, 15 mg/kg/week 8-CA plus 5 mg/kg/week hydroxychloroquine and 45 mg/kg/week 8-CA plus 5 mg/kg/week hydroxychloroquine. One million RMCCA-1 cells were subcutaneously injected in both flanks of each mouse and allowed to form tumors for 1 week. Therefore, 1 mouse would bear 2 tumors (one on each flank). The number of animals per group was calculated according to Dell et al. (23). A total of three mice per group (six tumors per group) were used. All treatments, 15 and 45 mg/kg 8-CA and 5 mg/kg hydroxychloroquine, were administered via intraperitoneal injection once a week. Hydroxychloroquine was administered on Tuesday and 8-CA was administered the day after. The treatments continued for 3 weeks or sooner if the mice had reached the humane endpoint. The human endpoint was reached when any of the following conditions was met i) weight loss 20% or more, or mouse did not eat, ii) mouse was unable to move, iii) mouse had open wound/necrosis/ulcerate, iv) presence of pain that interfered with regular behaviors, or v) the combination of the tumors longest side (left and right tumors) reached 20 mm. Mice which reached the humane endpoint or those reaching the end of the experiments were euthanized using carbon dioxide.

Tumor size was measured by using Vernier caliper and animals were weighed twice a week on Tuesday and Friday throughout each experiment. Tumor size was calculated by using the following formula. Tumor size=width (mm) × height (mm) × depth (mm) × 0.526. Tumors were excised and fixed in formalin for size comparison.

Statistical analysis. Data were plotted as mean±standard error of mean. Statistical analyses were performed using analysis of variance with Dunnett’s test or Student’s t-test, as appropriate. All experiments were performed at least in biological triplicate.

Results

8-CA reduced cholangiocarcinoma cell vitality and invasion. A common property of cancer therapeutic drugs is the reduction of cancer cell vitality. But this will be most beneficial when few or no effects are found on non-cancer cells, particularly fast-growing ones. MMNK-1, an immortalized non-cancer cholangiocyte cell line with a similar growth rate to that of cholangiocarcinoma cells, was used as a non-cancer control. Treatment for 1, 2 or 3 days with 8-CA at concentrations ranging from 0.1 μM to 100 μM were tested. The results showed that 8-CA reduced viability of all four cholangiocarcinoma cell lines tested, KKU-213, KKU-100, KKU-055 and RMCCA-1 (Figure 1A). However, KKU-055 was less responsive to 8-CA and its response was comparable to that of MMNK-1 cells (Figure 1A). Furthermore, DNA synthesis was evaluated by measuring the amount of BrdU incorporated into DNA strands. One-day treatment of 8-CA reduced BrdU incorporation to 73.8%, 71.5%, 88.3%, 79.6%, and 86.7 % in KKU-213, KKU-100, KKU-055, RMCCA-1 and MMNK-1 cells, respectively. Less inhibition of BrdU incorporation was observed in MMNK-1 and KKU-055 cells (Figure 1B).

Figure 1.
Figure 1.

8-Chloroadenosine (8-CA) reduced the cancerous characteristics of cholangiocarcinoma cells. A: 8-CA inhibited cell growth as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. B: Treatment with 10 μM 8-CA for 24 h reduced DNA synthesis. C: Treatment with 10 μM 8-chloroadenosine for 24 h reduced cholangiocarcinoma cell invasion through Matrigel. D: Treatment with 10 μM 8-CA for 24 h suppressed colony formation. E: Treatment with 10 μM 8-CA for 24 h increased the level of C/EBP homologous protein (CHOP) in cholangiocarcinoma cells but reduced it in cholangiocytes (MMNK-1). VC: Vehicle control. Statistical analysis of (A) was performed using analysis of variance with Dunnett’s test to compare the mean cell viability when treated with 100 μM of 8-CA; Student’s t-test was used for analysis of other measures. Significantly different at: *p<0.05, **p<0.01 and ***p<0.001; ns: not significantly different.

Another notable characteristic of cancer cells is that they can leave their original site and form colonies at distant sites during metastasis. To evaluate the effects of 8-CA on cancer metastasis, an invasion chamber and a colony-formation assay were used. Cholangiocarcinoma cells were tested for their ability to invade through Matrigel in the presence and absence of 10 μM 8-CA. The results showed that 8-CA reduced invading cells by 84.9%, 68.2%, 94.9% and 69.5% in KKU-213, KKU-100, KKU-055 and RMCCA-1, respectively (Figure 1C). MMNK-1 cells did not invade through Matrigel. In another experiment, cholangiocarcinoma cells were treated with either 10 μM 8-CA or the vehicle control (dimethyl sulfoxide) for 24 hours. Later, cells were washed, and 500 living cells were transferred to a 24-well plate in a fresh media without treatment. Cells were allowed to grow and form colonies in the new environment for 10 days. Compared to the vehicle control, 8-CA drastically reduced the number of colonies formed by 93.7%, 92.1%, 97.4% and 99.9% in KKU-213, KKU-100, KKU-055 and RMCCA-1 cells, respectively (Figure 1D).

8-CA caused endoplasmic reticulum (ER) stress and halted cells at the G2/M checkpoint. Previous results showed that 8-CA inhibited cell growth and reduced colony formation even with a single dose. We further investigated the mechanism underlying this phenomenon. CHOP is one of the commonly used markers of ER stress. The level of CHOP is up-regulated in response to ER stress and leads to apoptosis (24, 25). 8-CA at 10 μM caused ER stress, with the level of CHOP increasing significantly after 24 hours of treatment (Figure 1E). The CHOP level increased to 3.7-and 2.1-fold higher in KKU-213 and RMCCA-1 cells, respectively, compared to the vehicle control (Figure 1E). On the other hand, the CHOP level decreased dramatically in MMNK-1 cells when treated with 10 μM 8-CA (Figure 1E).

Cell-cycle arrest is expected when cells experience ER stress, therefore we determined the cell-cycle distribution in cholangiocarcinoma cells treated with 8-CA for 24 h. We found they were unable to pass the G2/M cell-cycle check point, resulting in significant increase of the cell population in the G2/M phase (Figure 2A). In contrast, the changes in populations of immortalized cholangiocytes, MMNK-1, in each phase after 8-CA treatment were subtler and not significant (Figure 2A).

Figure 2.
Figure 2.

8-Chloroadenosine (8-CA) caused cell-cycle arrest and induced apoptosis. A: Treatment with 10 μM 8-CA for 24 h halted cholangiocarcinoma cells at the G2/M cell-cycle check point. B: Treatment with 10 μM 8-CA for 24 h induced apoptotic cell death of cholangiocarcinoma cells. VC: Vehicle control. Statistical analysis was performed using Student’s t-test. Significantly different at: *p<0.05, **p<0.01, and ***p<0.001; ns: not significantly different.

8-CA induces apoptosis. Previous results demonstrated that 8-CA caused ER stress in cholangiocarcinoma cell lines and halted the cell cycle at the G2/M check point. In addition, colony formation showed that a single treatment for 24 h caused permanent damage and cells did not recover to form colonies even after 8-CA was removed. We further investigated the effects of 8-CA to elucidate the mechanism of cell death. 8-CA at 10 μM for 24 h increased the cholangiocarcinoma cell population undergoing apoptosis, both early and late apoptosis (Figure 2B). Early apoptosis increased from 2.3%, 5.5% and 5.7% to 16.6%, 20.1% and 12.5% in KKU-213, RMCCA-1 and MMNK-1, respectively. Similarly, late apoptosis also increased from 10.6%, 6.1% and 8.8% to 45.3%, 26.3% and 34.1% in KKU-213, RMCCA-1 and MMNK-1, respectively. However, the population of dead cells did not differ at this time point (Figure 2B).

Alterations in RNA and protein synthesis and mitochondrial function were not observed in the presence of 8-CA. Previous studies showed that 8-CA interfered with synthesis of RNA and protein and caused damage to mitochondria (26). However, treatment with 10 μM 8-CA for 24 h, the same time point at which apoptosis was observed, did not interfere with RNA or protein synthesis in cholangiocarcinoma cells (Figure 3A and B). The positive controls (actinomycin D for RNA synthesis and cycloheximide for protein synthesis), however, showed significant inhibition. In addition, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone was used to disrupt mitochondrial ATP synthesis and also to depolarize the mitochondrial membrane. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone reduced the mitochondria membrane potential significantly at both 4 and 24 hours of treatment in all cell lines tested. However, no change was observed in 8-CA-treated cells at these time points (Figure 3C). Effects of 8-CA in combination with chemotherapeutic drugs. There are some chemotherapeutic drugs that are currently used for cholangiocarcinoma treatment, including gemcitabine and cisplatin. Some other common drugs for cholangiocarcinoma treatment include 5-FU and paclitaxel. In addition, CX-4945 is a drug under clinical trial for cholangiocarcinoma treatment and was reported to induce a distinct cell death mechanism in cholangiocarcinoma cells (27). Combinations of 8-CA with the aforementioned drugs were examined. Single treatment with these chemotherapeutic drugs at 100 μM (Figure 4A and B) showed approximately similar levels of inhibition as compared to single treatment with 8-CA (Figure 1A). Unfortunately, the combination of 8-CA with these drugs for 24 hours did not provide better inhibition of KKU-213 (Figure 4A) or RMCCA-1 (Figure 4B) cholangiocarcinoma cells. Treatments with either single chemotherapeutic drugs or 8-CA at 100 μM inhibited cholangiocarcinoma cells with higher potency than the combination of the drugs with 8-CA (Figure 4A and B).

Figure 3.
Figure 3.

8-Chloroadenosine (8-CA) had no effect on the synthesis of biomolecules. RNA synthesis (A), protein synthesis (B) and mitochondrial membrane potential (C) were not altered by treatment with 10 μM 8-CA for 24 h. FCCP: Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, positive control; VC: vehicle control. Statistical analysis was performed using analysis of variance with Dunnett’s test. ***Significantly different at p<0.001.

Figure 4.
Figure 4.

Effects of the combination of 8-chloroadenosine (8-CA) with chemotherapeutic drugs cisplatin, paclitaxel, 5-fluorouracil (5-FU), CX-4945 and gemcitabine on cell viability. KKU-213 (A) and RMCCA-1 (B) cells were treated with a combination of 1 μM, 3 μM or 10 μM of 8-CA with other drugs for 24 h. Combinations provided no significantly greater inhibition than single treatment with either 8-CA or the chemotherapeutic drugs. Statistical analysis was performed using analysis of variance with Dunnett’s test.

Since combination with the aforementioned chemotherapeutic drugs did not provide additional inhibition of cholangiocarcinoma cell growth, a combination of 8-CA with autophagy inhibitors hydroxychloroquine and bafilomycin A1 was examined. A combination of these inhibitors with adenosine was reported to enhance the inhibitory effects of adenosine in cholangiocarcinoma cells (17). A combination of 100 μM 8-CA with 30 μM hydroxychloroquine for 24 hours yielded additional inhibition in RMCCA-1 cells. This combination reduced RMCCA-1 cell viability to 52.5%, while single treatment of 8-CA reduced it to only 73.0% (Figure 5A). Unfortunately, in KKU-213 cells, the combination of hydroxychloroquine with 8-CA for 24 hours did not show additional inhibition of cell growth (Figure 5A). However, hydroxychloroquine at 50 μM and bafilomycin A1 at both concentrations used did not have any additive effects. The additive effect of 8-CA and 30 μM hydroxychloroquine might not be due to inhibition of autophagy since 8-CA did not significantly increase the expression of the autophagy marker LC3 II (Figure 5B).

Figure 5.
Figure 5.

Effects of the combination of 8-chloroadenosine (8-CA) with autophagy inhibitors. Autophagy inhibitors were added 4 hours prior to the addition of 8-CA. A: Effect of a combination treatment with autophagy inhibitors, hydroxychloroquine (HCQ) (left) or bafilomycin A1 (Baf) (right) with 8-CA for 24 h. B: Level of autophagy marker microtubule-associated protein light chain 3 (LC3II) after 24 h of treatment with 10 μM 8-CA, alone and in combination with HCQ. Statistical analysis was performed using analysis of variance with Dunnett’s test to compare the mean cell viability when treated with 100 μM of 8-CA. Significantly different at: *p<0.05 and ***p<0.001.

To further validate results observed with in vitro experiments, an in vivo experiment was performed with 6-week-old male nude mice. RMCCA-1 cells were subcutaneously injected as described in the Materials and Methods section to form tumors. Treatment started 1 week after tumor injection when tumors had formed in all mice on both flanks, with an average size of 63.7±19.4 mm3. Hydroxychloroquine was chosen for the in vivo experiment because it was the only compound that showed an additive inhibitory effect when combined with 8-CA in the in vitro experiments. All treatments were performed using intraperitoneal injection. 8-CA was administered at either 15 mg/kg/week or 45 mg/kg/week with or without 5 mg/kg/week hydroxychloroquine. After 17 days of treatment, mice treated with 15 and 45 mg/kg/week 8-CA had tumor size of 221% and 357% as compared to the first day of treatment, while tumor size in the vehicle control group was 700% (Figure 6). The growth rate of tumors in mice treated with 15 and 45 mg/kg/week 8-CA were 10.1±0.9 mm3/day and 11.0±0.5 mm3/day, respectively, while that of the control group was 29.5±0.9 mm3/day (Figure 6B). Tumors in mice receiving 15 and 45 mg/kg/week 8-CA started to decrease in size after 10 days of treatment (Figure 6B). Tumor size and growth rate in these two groups were not statistically different. A combination of 8-CA with hydroxychloroquine achieved less inhibition compared to 8-CA alone.

Figure 6.
Figure 6.

8-Chloroadenosine (8-CA) inhibited tumor growth in vivo. All treatments, 15 and 45 mg/kg 8-CA and 5 mg/kg hydroxychloroquine (HCQ), were administered via intraperitoneal injection once a week for 3 weeks. Hydroxychloroquine was administered on Tuesday and 8-CA was administered the day after. A: Tumor size after 17 days of treatment. B: Progress of tumor growth during the experiment. HCQ: Hydroxychloroquine; VC: vehicle control. Statistical analysis of (B) was performed using analysis of variance with Dunnett’s test. *Significantly different at p<0.05; ns: not significant.

Discussion

The results show that 8-CA inhibits cholangiocarcinoma cells, with less effect on non-cancerous fast-growing cholangiocytes. 8-CA was reported to effectively inhibit leukemia stem cells (26). Interestingly, inhibitory effects of 8-CA on cholangiocarcinoma and leukemia stem cells appear to differ. 8-CA targets ribosomal RNA synthesis and mitochondrial metabolism in leukemia stem cells (26); however, 24-hour treatment with 10 μM 8-CA treatment did not alter these processes in cholangiocarcinoma cells (Figure 3). Moreover, 8-CA did not alter DNA synthesis in leukemia stem cells (26), but significantly inhibited it in cholangiocarcinoma cells (Figure 1A). A previous study reported that 8-CA depleted endogenous ATP and induced autophagic cell death in breast cancer cells (28). On the other hand, 8-CA did not induce autophagy in cholangiocarcinoma cell, but instead induced ER stress and apoptosis (Figure 1E and Figure 2). A similar result was reported in coronary artery endothelial cells (29). Although ER stress was not a common criterion in stopping cells at the G2/M checkpoint during the cell cycle, 8-CA was recently shown to promote G2/M cell-cycle arrest in lung cancer cells and mouse embryogenic fibroblasts (30, 31). This arrest was due to ER stress caused by a prolonged unfolded protein response (31).

Multi-drug treatment is common for cancer. To our knowledge, this is the first study to combine 8-CA with current cholangiocarcinoma chemotherapeutic drugs, namely gemcitabine, cisplatin, paclitaxel, 5-FU, and CX-4945. Unfortunately, the combinations did not provide additive inhibition of cholangiocarcinoma cell growth. The results suggest that treatment with 8-CA alone at 10 μM has the same or a greater inhibitory effect on cholangiocarcinoma than its combination with chemotherapeutic drugs or using these drugs alone (Figure 4). A combination of 100 μM 8-CA with 30 μM hydroxychloroquine, an autophagy inhibitor, enhanced the inhibitory effect on RMCCA-1 cells (Figure 5A). However, the combination of 8-CA with bafilomycin A1, another autophagy inhibitor, failed to enhance the inhibitory effect of 8-CA (Figure 5A). It would also be interesting to investigate the effect of hydroxychloroquine on cholangiocarcinoma cells since our results suggest that the additive effect of 8-CA and hydroxychloroquine observed in RMCCA-1 was not from autophagy inhibition.

Hydroxychloroquine is metabolized by cytochrome P450 (CYP450), a hemeprotein having a key role in drug metabolism, producing pharmacologically active form, such as des-ethyl chloroquine and bis-des-ethyl chloroquine (32-34). A change in the enzymatic activity of CYP450 isoenzymes might be a result of hydroxychloroquine administration because hydroxychloroquine and its metabolites were recently shown to inhibit CYP450 2D6 and CYP450 3A (35). In addition, chloroquine is reported to be an inducer of multidrug resistance-associated ATP-binding cassette subfamily C member 1, and hydroxychloroquine is reported to inhibit organic anion transporting polypeptide 1A2 (34). Although we are not aware of any report on interference of hydroxychloroquine with equilibrative nucleoside transporters (ENT), chloroquine and hydroxychloroquine were shown to interfere with some drug transporters as mentioned above (32, 34). Therefore, interference of hydroxychloroquine with 8-CA transportation via ENT1 is a plausible mechanism. ENT1 is important for adenosine-induced inhibitory effects in CCA cells. We recently reported that ENT1 is a transporter adenosine uses to enter cholangiocarcinoma cells and exerts its inhibitory effects (36).

Although there is currently no direct evidence showing that CYP450 metabolizes adenosine or 8-CA, there is a report demonstrating that CYP450 1B1, isoenzyme is responsible for gemcitabine resistance in pancreatic cancer (37). Since both gemcitabine and 8-CA are nucleoside analogs and share similar inhibitory mechanisms, 8-CA activity might be interfered with by activation or inhibition of one of the CYP450 isoenzymes.

Further study on 8-CA metabolism in liver and its interaction with CYP450 will provide more information on how to combine this compound effectively with other drugs to maximize the usage of 8-CA in patients with cholangio-carcinoma.

Acknowledgements

This project was supported by Thailand Science Research and Innovation (TSRI) and Chulabhorn Research Institute (Grant No. 3681/4274359).

Footnotes

  • Authors’ Contributions

    Conceptualization: J.L., J. Svasti, and J. Satayavivad. Methodology: J.L. and J. Satayavivad. Validation: J.L., J. Svasti, and J. Satayavivad. Formal analysis: J.L. Investigation: J.L. Resources: J. Satayavivad. Writing – original draft preparation: J.L. Writing – review and editing: J.L., J. Svasti, and J. Satayavivad. Supervision: J. Svasti and J. Satayavivad. Funding acquisition: J. Satayavivad. All Authors read and approved the final article.

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest.

  • Received July 18, 2023.
  • Revision received October 12, 2023.
  • Accepted October 16, 2023.

References

  1. ↵
    1. Sithithaworn P,
    2. Yongvanit P,
    3. Duenngai K,
    4. Kiatsopit N,
    5. Pairojkul C
    : Roles of liver fluke infection as risk factor for cholangiocarcinoma. J Hepatobiliary Pancreat Sci 21(5): 301-308, 2014. DOI: 10.1002/jhbp.62
    OpenUrlCrossRefPubMed
    1. Zhang H,
    2. Yang T,
    3. Wu M,
    4. Shen F
    : Intrahepatic cholangiocarcinoma: Epidemiology, risk factors, diagnosis and surgical management. Cancer Lett 379(2): 198-205, 2016. DOI: 10.1016/j.canlet.2015.09.008
    OpenUrlCrossRefPubMed
    1. Banales JM,
    2. Cardinale V,
    3. Carpino G,
    4. Marzioni M,
    5. Andersen JB,
    6. Invernizzi P,
    7. Lind GE,
    8. Folseraas T,
    9. Forbes SJ,
    10. Fouassier L,
    11. Geier A,
    12. Calvisi DF,
    13. Mertens JC,
    14. Trauner M,
    15. Benedetti A,
    16. Maroni L,
    17. Vaquero J,
    18. Macias RIR,
    19. Raggi C,
    20. Perugorria MJ,
    21. Gaudio E,
    22. Boberg KM,
    23. Marin JJG,
    24. Alvaro D
    : Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Nat Rev Gastroenterol Hepatol 13(5): 261-280, 2016. DOI: 10.1038/nrgastro.2016.51
    OpenUrlCrossRefPubMed
  4. ↵
    1. Valle JW,
    2. Kelley RK,
    3. Nervi B,
    4. Oh D,
    5. Zhu AX
    : Biliary tract cancer. Lancet 397(10272): 428-444, 2021. DOI: 10.1016/S0140-6736(21)00153-7
    OpenUrlCrossRefPubMed
  5. ↵
    1. Sangrajrang S,
    2. Laversanne M,
    3. Bausom R,
    4. Mery L,
    5. Bray F
    : Cancer incidence and cancer control in Bangkok, Thailand: Results from the cancer registry 2011-15 and projections to 2035. Cancer Epidemiol 67: 101765, 2020. DOI: 10.1016/j.canep.2020.101765
    OpenUrlCrossRef
  6. ↵
    1. Elvevi A,
    2. Laffusa A,
    3. Scaravaglio M,
    4. Rossi RE,
    5. Longarini R,
    6. Stagno AM,
    7. Cristoferi L,
    8. Ciaccio A,
    9. Cortinovis DL,
    10. Invernizzi P,
    11. Massironi S
    : Clinical treatment of cholangiocarcinoma: an updated comprehensive review. Ann Hepatol 27(5): 100737, 2022. DOI: 10.1016/j.aohep.2022.100737
    OpenUrlCrossRefPubMed
    1. Yao J,
    2. Liang X,
    3. Liu Y,
    4. Li S,
    5. Zheng M
    : Trends in incidence and prognostic factors of two subtypes of primary liver cancers: a surveillance, epidemiology, and end results-based population study. Cancer Control 29: 10732748211051548, 2022. DOI: 10.1177/10732748211051548
    OpenUrlCrossRefPubMed
  8. ↵
    1. Banales JM,
    2. Marin JJG,
    3. Lamarca A,
    4. Rodrigues PM,
    5. Khan SA,
    6. Roberts LR,
    7. Cardinale V,
    8. Carpino G,
    9. Andersen JB,
    10. Braconi C,
    11. Calvisi DF,
    12. Perugorria MJ,
    13. Fabris L,
    14. Boulter L,
    15. Macias RIR,
    16. Gaudio E,
    17. Alvaro D,
    18. Gradilone SA,
    19. Strazzabosco M,
    20. Marzioni M,
    21. Coulouarn C,
    22. Fouassier L,
    23. Raggi C,
    24. Invernizzi P,
    25. Mertens JC,
    26. Moncsek A,
    27. Rizvi S,
    28. Heimbach J,
    29. Koerkamp BG,
    30. Bruix J,
    31. Forner A,
    32. Bridgewater J,
    33. Valle JW,
    34. Gores GJ
    : Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol 17(9): 557-588, 2020. DOI: 10.1038/s41575-020-0310-z
    OpenUrlCrossRefPubMed
    1. Khan AS,
    2. Dageforde LA
    : Cholangiocarcinoma. Surg Clin North Am 99(2): 315-335, 2019. DOI: 10.1016/j.suc.2018.12.004
    OpenUrlCrossRefPubMed
  10. ↵
    1. Brindley PJ,
    2. Bachini M,
    3. Ilyas SI,
    4. Khan SA,
    5. Loukas A,
    6. Sirica AE,
    7. Teh BT,
    8. Wongkham S,
    9. Gores GJ
    : Cholangiocarcinoma. Nat Rev Dis Primers 7(1): 65, 2021. DOI: 10.1038/s41572-021-00300-2
    OpenUrlCrossRefPubMed
  11. ↵
    1. Kamsa-ard S,
    2. Kamsa-ard S,
    3. Luvira V,
    4. Suwanrungruang K,
    5. Vatanasapt P,
    6. Wiangnon S
    : Risk factors for cholangiocarcinoma in Thailand: a systematic review and meta-analysis. Asian Pac J Cancer Prev 19(3): 605-614, 2018. DOI: 10.22034/APJCP.2018.19.3.605
    OpenUrlCrossRefPubMed
  12. ↵
    1. Cillo U,
    2. Fondevila C,
    3. Donadon M,
    4. Gringeri E,
    5. Mocchegiani F,
    6. Schlitt HJ,
    7. Ijzermans JNM,
    8. Vivarelli M,
    9. Zieniewicz K,
    10. Olde Damink SWM,
    11. Groot Koerkamp B
    : Surgery for cholangiocarcinoma. Liver Int 39 Suppl 1(Suppl Suppl 1): 143-155, 2019. DOI: 10.1111/liv.14089
    OpenUrlCrossRefPubMed
  13. ↵
    1. Oliverius M,
    2. Havluj L,
    3. Hajer J,
    4. Soumarova R,
    5. Hrudka J,
    6. Gurlich R
    : Surgery for cholangiocarcinoma. Cas Lek Cesk 158(2): 73-77, 2019.
    OpenUrlPubMed
  14. ↵
    1. Chaiteerakij R,
    2. Harmsen WS,
    3. Marrero CR,
    4. Aboelsoud MM,
    5. Ndzengue A,
    6. Kaiya J,
    7. Therneau TM,
    8. Sanchez W,
    9. Gores GJ,
    10. Roberts LR
    : A new clinically based staging system for perihilar cholangiocarcinoma. Am J Gastroenterol 109(12): 1881-1890, 2014. DOI: 10.1038/ajg.2014.327
    OpenUrlCrossRefPubMed
  15. ↵
    1. Oh DY,
    2. Lee KH,
    3. Lee DW,
    4. Yoon J,
    5. Kim TY,
    6. Bang JH,
    7. Nam AR,
    8. Oh KS,
    9. Kim JM,
    10. Lee Y,
    11. Guthrie V,
    12. McCoon P,
    13. Li W,
    14. Wu S,
    15. Zhang Q,
    16. Rebelatto MC,
    17. Kim JW
    : Gemcitabine and cisplatin plus durvalumab with or without tremelimumab in chemotherapy-naive patients with advanced biliary tract cancer: an open-label, single-centre, phase 2 study. Lancet Gastroenterol Hepatol 7(6): 522-532, 2022. DOI: 10.1016/S2468-1253(22)00043-7
    OpenUrlCrossRefPubMed
  16. ↵
    1. Lertsuwan J,
    2. Ruchirawat M
    : Inhibitory effects of ATP and adenosine on cholangiocarcinoma cell proliferation and motility. Anticancer Res 37(7): 3553-3561, 2017. DOI: 10.21873/anticanres.11725
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Lertsuwan J,
    2. Svasti J,
    3. Satayavivad J
    : Adenosine induces autophagy in cholangiocarcinoma cells. Anticancer Res 41(8): 3769-3778, 2021. DOI: 10.21873/anticanres.15169
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Panjehpour M,
    2. Karami-Tehrani F
    : Adenosine modulates cell growth in the human breast cancer cells via adenosine receptors. Oncol Res 16(12): 575-585, 2007. DOI: 10.3727/000000007783629981
    OpenUrlCrossRefPubMed
  19. ↵
    1. Lertsuwan K,
    2. Peters W,
    3. Johnson L,
    4. Lertsuwan J,
    5. Marwa I,
    6. Sikes RA
    : Purinergic receptor expression and cellular responses to purinergic agonists in human prostate cancer cells. Anticancer Res 37(2): 529-538, 2017. DOI: 10.21873/anticanres.11345
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Ding HY,
    2. Yang WY,
    3. Zhang LH,
    4. Li L,
    5. Xie F,
    6. Li HY,
    7. Chen XY,
    8. Tu Z,
    9. Li Y,
    10. Chen Y,
    11. Yang SY
    : 8-chloro-adenosine inhibits proliferation of MDA-MB-231 and SK-BR-3 breast cancer cells by regulating ADAR1/p53 signaling pathway. Cell Transplant 29: 963689720958656, 2020. DOI: 10.1177/0963689720958656
    OpenUrlCrossRefPubMed
  21. ↵
    1. Buettner R,
    2. Nguyen LXT,
    3. Morales C,
    4. Chen MH,
    5. Wu X,
    6. Chen LS,
    7. Hoang DH,
    8. Hernandez Vargas S,
    9. Pullarkat V,
    10. Gandhi V,
    11. Marcucci G,
    12. Rosen ST
    : Targeting the metabolic vulnerability of acute myeloid leukemia blasts with a combination of venetoclax and 8-chloro-adenosine. J Hematol Oncol 14(1): 70, 2021. DOI: 10.1186/s13045-021-01076-4
    OpenUrlCrossRefPubMed
  22. ↵
    1. Rattanasinganchan P,
    2. Leelawat K,
    3. Treepongkaruna SA,
    4. Tocharoentanaphol C,
    5. Subwongcharoen S,
    6. Suthiphongchai T,
    7. Tohtong R
    : Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient. World J Gastroenterol 12(40): 6500-6506, 2006. DOI: 10.3748/wjg.v12.i40.6500
    OpenUrlCrossRefPubMed
  23. ↵
    1. Dell RB,
    2. Holleran S,
    3. Ramakrishnan R
    : Sample size determination. ILAR J 43(4): 207-213, 2002. DOI: 10.1093/ilar.43.4.207
    OpenUrlCrossRefPubMed
  24. ↵
    1. Hu H,
    2. Tian M,
    3. Ding C,
    4. Yu S
    : The C/EBP homologous protein (CHOP) transcription factor functions in endoplasmic reticulum stress-induced apoptosis and microbial infection. Front Immunol 9: 3083, 2019. DOI: 10.3389/fimmu.2018.03083
    OpenUrlCrossRefPubMed
  25. ↵
    1. Oyadomari S,
    2. Mori M
    : Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11(4): 381-389, 2004. DOI: 10.1038/sj.cdd.4401373
    OpenUrlCrossRefPubMed
  26. ↵
    1. Hoang DH,
    2. Morales C,
    3. Rodriguez IR,
    4. Valerio M,
    5. Guo J,
    6. Chen MH,
    7. Wu X,
    8. Horne D,
    9. Gandhi V,
    10. Chen LS,
    11. Zhang B,
    12. Pullarkat V,
    13. Rosen ST,
    14. Marcucci G,
    15. Buettner R,
    16. Nguyen LXT
    : Synergy of Venetoclax and 8-chloro-adenosine in AML: The interplay of rRNA inhibition and fatty acid metabolism. Cancers (Basel) 14(6): 1446, 2022. DOI: 10.3390/cancers14061446
    OpenUrlCrossRefPubMed
  27. ↵
    1. Lertsuwan J,
    2. Lertsuwan K,
    3. Sawasdichai A,
    4. Tasnawijitwong N,
    5. Lee KY,
    6. Kitchen P,
    7. Afford S,
    8. Gaston K,
    9. Jayaraman PS,
    10. Satayavivad J
    : CX-4945 induces methuosis in cholangiocarcinoma cell lines by a CK2-independent mechanism. Cancers (Basel) 10(9): 283, 2018. DOI: 10.3390/cancers10090283
    OpenUrlCrossRefPubMed
  28. ↵
    1. Stellrecht CM,
    2. Vangapandu HV,
    3. Le XF,
    4. Mao W,
    5. Shentu S
    : ATP directed agent, 8-chloro-adenosine, induces AMP activated protein kinase activity, leading to autophagic cell death in breast cancer cells. J Hematol Oncol 7: 23, 2014. DOI: 10.1186/1756-8722-7-23
    OpenUrlCrossRefPubMed
  29. ↵
    1. Tang V,
    2. Fu S,
    3. Rayner BS,
    4. Hawkins CL
    : 8-Chloroadenosine induces apoptosis in human coronary artery endothelial cells through the activation of the unfolded protein response. Redox Biol 26: 101274, 2019. DOI: 10.1016/j.redox.2019.101274
    OpenUrlCrossRefPubMed
  30. ↵
    1. Duan HY,
    2. Cao JX,
    3. Qi JJ,
    4. Wu GS,
    5. Li SY,
    6. An GS,
    7. Jia HT,
    8. Cai WW,
    9. Ni JH
    : E2F1 enhances 8-Chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells. Biochemistry 77(3): 261-269, 2012. DOI: 10.1134/S0006297912030042
    OpenUrlCrossRefPubMed
  31. ↵
    1. Lee D,
    2. Hokinson D,
    3. Park S,
    4. Elvira R,
    5. Kusuma F,
    6. Lee JM,
    7. Yun M,
    8. Lee SG,
    9. Han J
    : ER stress induces cell cycle arrest at the G2/M phase through eIF2α phosphorylation and GADD45α. Int J Mol Sci 20(24): 6309, 2019. DOI: 10.3390/ijms20246309
    OpenUrlCrossRefPubMed
  32. ↵
    1. Charlier B,
    2. Pingeon M,
    3. Dal Piaz F,
    4. Conti V,
    5. Valentini G,
    6. Filippelli A,
    7. Izzo V
    : Development of a novel ion-pairing HPLC-FL method for the separation and quantification of hydroxychloroquine and its metabolites in whole blood. Biomed Chromatogr 32(8): e4258, 2018. DOI: 10.1002/bmc.4258
    OpenUrlCrossRefPubMed
    1. Lee JY,
    2. Vinayagamoorthy N,
    3. Han K,
    4. Kwok SK,
    5. Ju JH,
    6. Park KS,
    7. Jung SH,
    8. Park SW,
    9. Chung YJ,
    10. Park SH
    : Association of polymorphisms of cytochrome P450 2D6 with blood hydroxychloroquine levels in patients with systemic lupus erythematosus. Arthritis Rheumatol 68(1): 184-190, 2016. DOI: 10.1002/art.39402
    OpenUrlCrossRefPubMed
  34. ↵
    1. Rendic S,
    2. Guengerich FP
    : Metabolism and interactions of chloroquine and hydroxychloroquine with human cytochrome P450 enzymes and drug transporters. Curr Drug Metab 21(14): 1127-1135, 2020. DOI: 10.2174/1389200221999201208211537
    OpenUrlCrossRefPubMed
  35. ↵
    1. Paludetto MN,
    2. Kurkela M,
    3. Kahma H,
    4. Backman JT,
    5. Niemi M,
    6. Filppula AM
    : Hydroxychloroquine is metabolized by cytochrome P450 2D6, 3A4, and 2C8, and inhibits cytochrome P450 2D6, while its metabolites also inhibit cytochrome P450 3A in vitro. Drug Metab Dispos 51(3): 293-305, 2023. DOI: 10.1124/dmd.122.001018
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Lertsuwan K,
    2. Phoaubon S,
    3. Tasnawijitwong N,
    4. Lertsuwan J
    : Adenosine suppresses cholangiocarcinoma cell growth and invasion in equilibrative nucleoside transporters-dependent pathway. Int J Mol Sci 21(3): 814, 2020. DOI: 10.3390/ijms21030814
    OpenUrlCrossRefPubMed
  37. ↵
    1. Yada E,
    2. Kasajima R,
    3. Niida A,
    4. Imoto S,
    5. Miyano S,
    6. Miyagi Y,
    7. Sasada T,
    8. Wada S
    : Possible role of cytochrome P450 1B1 in the mechanism of gemcitabine resistance in pancreatic cancer. Biomedicines 9(10): 1396, 2021. DOI: 10.3390/biomedicines9101396
    OpenUrlCrossRefPubMed
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8-Chloroadenosine Induces ER Stress and Apoptotic Cell Death in Cholangiocarcinoma Cells
JOMNARONG LERTSUWAN, JISNUSON SVASTI, JUTAMAAD SATAYAVIVAD
Anticancer Research Dec 2023, 43 (12) 5425-5436; DOI: 10.21873/anticanres.16746
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