Abstract
Background/Aim: Anti-apoptotic proteins, including Bcl-2 and Bcl-xL, hinder cancer treatment, and several drugs targeting these molecules have been developed. One is ABT-263 (navitoclax), which targets Bcl-2, Bcl-xL, and Bcl-w. On the other hand, hydroxychloroquine (HCQ) has been used as a drug for malaria infection and autoimmune disease. HCQ can exert a similar effect as chloroquine with fewer adverse events. In addition, HCQ exerts antitumor activity. In the present study, the effects of HCQ on ABT-263-induced antitumor activities were examined using three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3). Materials and Methods: In vitro effects of HCQ and ABT-263 were examined by cell viability, colony-forming assays, and flow cytometry. Protein expression was determined by immunoblotting. In vivo effects of HCQ and ABT-263 were examined by a xenograft mice model. Results: Combined treatment with HCQ and ABT-263 synergistically decreased the viability of only BxPC-3 cells. This synergistic effect was not observed when HCQ was combined with ABT-199, an inhibitor specific to Bcl-2. The combination of HCQ and ABT-263 induced caspase-dependent apoptosis. Protein expression of Bcl-xL was more highly expressed in BxPC-3 cells than in the other two cell lines, and the combination of HCQ with a Bcl-xL inhibitor or siRNA-mediated knockdown of Bcl-xL induced apoptosis in BxPC-3 cells. Combination therapy with HCQ and ABT 737, an ABT-263 analogue, suppressed the in vivo growth of BxPC-3 with transient body-weight loss. Conclusion: HCQ effectively promotes Bcl-xL inhibition-induced apoptosis in BxPC-3 human pancreatic cancer cells.
The goal of anticancer therapy is to induce cancer cell death without damage to normal cells and tissues. However, cancer cells evade therapies via various mechanisms. One major mechanism is resistance to apoptosis, mainly via increased expression of anti-apoptotic proteins. Although numerous molecules are involved in cell death (1), Bcl-2 family proteins play crucial roles in resistance to apoptosis by cancer cells (2). Bcl-2 family proteins consist of anti-apoptotic proteins, including Bcl-2, Bcl-xL, Bcl-w, and Mcl-1, and pro-apoptotic proteins, including Bax and Bak (3). Anti-apoptotic proteins inhibit cell death by sequestering pro-apoptotic proteins and preventing their oligomerization (4-6). Therefore, anti-apoptotic Bcl-2 family proteins have been suggested as promising targets in treating cancers and several molecular-targeting drugs have been developed. One is ABT-263, an orally bioavailable Bcl-2 family inhibitor against Bcl-2, Bcl-xL, and Bcl-w (7, 8), which has been applied clinically (9). We previously reported that ABT-263, and its analogue ABT-737 with the same specificity, can enhance drug-induced antitumor effects against human prostate and pancreatic cancer cells via Bcl-xL inhibition (10, 11). Targeting Bcl-xL may augment the antitumor effects of other drugs.
Hydroxychloroquine (HCQ) has been used for the treatment of malaria and autoimmune diseases (12). Chloroquine (CQ) and HCQ belong to the quinolone family and show similar pharmacological effects. However, HCQ is more useful than CQ for clinical applications because CQ frequently involves severe ocular and gastrointestinal adverse effects (12). Furthermore, both CQ and HCQ have the potential to exert antitumor activity (13). However, because HCQ is less toxic than CQ, HCQ was expected to be a better anticancer drug. HCQ has been studied in many cancer types, especially as an adjuvant agent to increase the therapeutic efficacy or overcome therapy resistance (14-16).
Pancreatic cancer is an aggressive malignancy with a high metastatic risk and low survival rate compared to other cancers (17). Its diagnosis and treatment are challenging (18), and new treatment modalities are required to improve its prognosis. Therefore, in the present study, we investigated the combination effect of HCQ with Bcl-2 family inhibitors, including ABT-263 and ABT-199, using three human pancreatic cancer cell lines.
Materials and Methods
Cell lines and reagents. Three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3) were kindly provided by Dr. K. Takenaga (Shimane University Faculty of Medicine) and were maintained in Dulbecco’s modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) fetal calf serum (Invitrogen, Grand Island, NY, USA) and 20 μg/ml of gentamicin (Sigma-Aldrich). HCQ with >98% purity was purchased from InvivoGen (San Diego, CA, USA) and diluted in saline. ABT-263 and ABT-737 were purchased from Active Biochemicals Co., Ltd. (Wan Chai, Hong Kong). ABT-199 was purchased from ChemieTek (Indianapolis, IN, USA). A1331852 was purchased from Selleck (Houston, TX, USA). These reagents were >98% purity and diluted in DMSO. Pan-caspase inhibitor (Z-VAD-FMK) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Both caspase-8 inhibitor (Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) were purchased from R&D Systems (Minneapolis, MN, USA). These caspase reagents were diluted in DMSO.
Cell viability assay. Cell viability was measured using the WST-8 assay (Nacalai Tesque, Kyoto, Japan). Cancer cells (2-4×104 cells/well) were seeded in a volume of 100 μl in 96-well flat-bottom plates. The indicated doses of the reagents were added at the initiation of culture. The tested maximum doses of HCQ, ABT-263, and ABT-199 were 100 μM, 40 μM, and 40 μM, respectively. Based on IC50, the doses of reagents were determined for subsequent experiments. Three days later, 10 μl of WST-8 solution was added to each well and the plates were incubated for an additional 3 h. The absorbance at 450 nm was measured using a microplate reader (Beckman Coulter, Brea, CA, USA). Whether the effects were synergistic or additive was determined using the normalized isobologram.
Colony-forming assay. Cells were seeded into 6-well flat-bottom plates and cultured with the indicated doses of HCQ for 12-16 days. Next, to visualize colonies, the cells were fixed in methanol, stained with 0.05% (w/v) crystal violet, and counted.
Immunoblotting. Cells were lysed using a mammalian protein extraction reagent (M-PER; Thermo Scientific, Rockford, IL, USA) containing a protease inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4-12% (w/v) gradient or 12% (w/v) SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked and then incubated with the following primary antibodies: anti-Bcl-2 [clone 100, #658701 (×1,000): BioLegend, San Diego, CA, USA], anti-Bcl-xL [clone 54H6, #2764S (×1,000): Cell Signaling Technology (CST), Danvers, MA, USA], or anti-β-actin [polyclonal 6221, #622102 (×1,000): BioLegend]. After washing, the membranes were incubated at room temperature for 30 min with either goat antirabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Invitrogen). Protein bands were visualized using the CDP-star chemiluminescence technique and imaged using an Image Quant LAS-4000 system (Fuji Film, Tokyo, Japan). The band intensities were scanned and quantified using the ImageJ software (http://rsb.info.nih.gov/ij/).
siRNA transfection. siRNA transfection was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. The following siRNAs were used: Bcl-2 siRNA (#6516; CST), Bcl-xL siRNA (#6363; CST), and control (scramble) siRNA (#6568; CST). The transfected cancer cells were used for immunoblotting and apoptosis experiments at 3 and 2 days after siRNA transfection, respectively.
Flow cytometric analysis. Cancer cells (4×104/well) were seeded in a volume of 1 ml in 24-well plates with HCQ (10 μM) and/or ABT-263 (5 μM). Two days later, the cultured cells were harvested and stained with FITC-conjugated annexin V (AV) and propidium iodide (PI) (BioVision, Mountain View, CA, USA). Then, the cells were analyzed using CytoFLEX (Beckman Coulter) or FACSCalibur (Japan BD Biosciences, Tokyo, Japan).
In vivo xenograft model. Female BALB nu/nu mice (CLEA Japan), aged 5-6 weeks, were kept under pathogen-free conditions and fed freely. Mice were inoculated in the right flank with 2×106 BxPC-3 cells and Matrigel (Japan BD Biosciences) at a 1:1 volume ratio in a total volume of 100 μl. When the tumor diameter reached approximately 5-6 mm, the mice were pooled and divided into four groups. Each group consisted of six mice. HCQ (60 mg/kg) was intraperitoneally administered on days 0, 1, 3, 4, 6, 7, 9, 10, 12, and 13 after grouping. ABT-737 (50 mg/kg) was administered intraperitoneally on days 2, 5, 8, 11, and 14 after grouping. Saline and DMSO were administered as vehicle controls for the HCQ and ABT-737 treatments, respectively, in the same volumes. The tumor sizes were measured using calipers. The tumor size and body weights were monitored twice weekly. However, mice were checked every day from the treatment-starting day until 7 days after the last treatment, i.e., day 21 after starting the treatment. The tumor volume was calculated as follows: tumor volume (mm3)=(length × width2) ÷ 2. On day 25 after starting the treatment, all mice were euthanized by using carbon dioxide; CO2 was displaced in10 sec. The criteria for euthanasia were determined when the tumor diameter reached at 10 mm. All experiments with animals in this study were approved by the Animal Care and Use Committee of Shimane University (IZ3-94).
Statistical analysis. Data were analyzed using the unpaired two-tailed Student’s t-test (between two groups) or an analysis of variance (ANOVA) with Tukey–Kramer test (for more than two groups). p- Values <0.05 were considered indicative of statistical significance.
Results
HCQ with ABT-263, but not ABT-199, effectively decreased BxPC-3 cell viability. Initially, the effects of HCQ on the in vitro growth of three human pancreatic cancer cell lines, PANC-1, MiaPaCa-2, and BxPC-3, were examined (Figure 1A). HCQ significantly reduced the viability of three cell lines in a dose-dependent manner. The IC50 values of HCQ in these three cell lines were 30.5 μM, 25.0 μM, and 17.5 μM, respectively. In addition, the effects of HCQ on the colony-forming ability of cancer cells were examined. The results show that HCQ similarly inhibited the colony number in the three cell lines (Figure 1B). Figure 1C shows the representative results. In the colony-forming assay, BxPC-3 cells were more sensitive to HCQ than the other two cell lines.
Effects of HCQ on growth of three human pancreatic cancer cell lines. (A) Three human pancreatic cancer lines were cultured with HCQ at the indicated doses. After 72 h, the cell viabilities were determined using the WST-8 assay. The data represent the mean values±SD; statistical difference was evaluated by Student’s t-test (*p<0.05, **p<0.01 compared with the untreated group). The experiments were performed in triplicate and repeated thrice. (B) Three cell lines were cultured in the presence of the indicated doses of HCQ. After 12-16 days, colonies were fixed with methanol, stained with crystal violet, and counted. The experiments were performed in triplicate and repeated thrice. (C) Representative data from the colony forming assays are shown. The data represent the mean values±SD; statistical difference was evaluated by Student’s t-test (**p<0.01 compared to the untreated group). HCQ, Hydroxychloroquine.
Next, the sensitivity of cancer cells to ABT-263 and ABT-199 was examined. The IC50 values of ABT-263 in PANC-1, MiaPaCa-2, and BxPC-3 cell lines were 14.0 μM, 16.0 μM, and 8.0 μM, respectively, while that of ABT-199 in MiaPaCa-2 and BxPC-3 cell lines was 18.0 μM. The IC50 of ABT-199 in PANC-1 cells was greater than 40 μM. To this end, suboptimal doses were used for the subsequent experiments to test the combination effects of 10, 20, and 30 μM HCQ with 2.5, 5, and 10 μM ABT-263 or ABT-199. As a result, when BxPC-3 cells were cultured with HCQ (10 and 20 μM) and ABT-263 (2.5, 5, and 10 μM), their viability decreased more profoundly compared to the combination of HCQ and ABT-199 (Figure 2A). Unexpectedly, ABT-199 increased PANC-1 cell viability with or without 10 μM HCQ. In contrast, ABT-199 decreased MiaPaCa-2 cell viability with or without 10 μM HCQ. Representative results for the BxPC-3 cells are shown in Figure 2B and C. The combination of ABT-263 (5 μM) and HCQ (10 μM) effectively decreased cell viability compared to either treatment alone, and this effect was synergistic based on the normalized isobologram. On the other hand, ABT-199 could not enhance the antitumor effect of HCQ on BxPC-3 cells. To this end, BxPC-3 cell line was the only one taken forward for experiments.
Combined effect of HCQ and ABT-263 on BxPC-3 cells. (A) Three cell lines were cultured with the indicated doses of HCQ with ABT-263 or ABT-199. After 72 h, the cell viabilities were determined using the WST-8 assay. The data represent the means±SD, analyzed using the Student’s t-test (*p<0.05, **p<0.01 compared to the no inhibitor group). The experiments were performed in triplicate and repeated thrice. (B, C) Representative results for HCQ and ABT-263/ABT-199 treatment are shown. The data represent the means+SD, analyzed using ANOVA with the Tukey–Kramer test (**p<0.01, N.S.: non-significant). Whether the effects were synergistic or additive was evaluated using the normalized isobologram. The experiments were performed thrice. HCQ: Hydroxychloroquine.
HCQ plus ABT-263 promoted apoptosis in BxPC-3 cells. Next, given that cell viability reflects both cell growth and death, whether the cell death was due to apoptosis was investigated. Apoptosis assay was performed on day 2 because apoptosis was evident at this timing. An apoptosis assay using annexin V and PI showed that combination of HCQ and ABT-263 significantly increased the percentages of annexin V+ PI– early apoptotic cells compared with monotherapy (Figure 3A). A statistical difference was not observed regarding annexin V+ PI+ late apoptosis between ABT-263 alone and ABT-263 combined with HCQ. Representative results are shown in Figure 3B.
Caspase-dependent apoptosis in BxPC-3 cells treated with both HCQ and ABT-263. Next, whether apoptosis in treated BxPC-3 cells was caspase-dependent was further investigated. The addition of the pan-caspase inhibitor Z-VAD significantly decreased the percentage of annexin V+ PI-early apoptotic BxPC-3 cells (Figure 3C). The addition of either Z-IETD (caspase-8 inhibitor) or Z-LEHD (caspase-9 inhibitor) also significantly inhibited the induction of early apoptosis. Representative results are shown in Figure 3D. A statistical difference was not observed regarding annexin V+ PI+ late apoptosis of BxPC-3 cells. These results indicate that the BxPC-3 cell death induced by a combination of HCQ and ABT-263 was caspase-dependent apoptosis and that both caspase-8 and caspase-9 were involved in the apoptosis.
Caspase-dependent apoptosis in BxPC-3 cells treated with both HCQ and ABT-263. (A) BxPC-3 cells treated with HCQ (10 μM) and ABT-263 (2.5 μM) in the presence of caspase inhibitors for 48 h. After staining with AV-FITC and PI, cells were analyzed using flow cytometry. The data represent the mean values±SD; statistical difference was evaluated by ANOVA with Tukey–Kramer test (*p<0.05, **p<0.01, N.S., not significant). The experiments were performed in triplicate and repeated twice. Z-VAD, pan-caspase inhibitor; Z-IETD, caspase-8 inhibitor; Z-LEHD, caspase-9 inhibitor. The same volume of DMSO was added as a vehicle control. (B) Representative results are shown; numbers represent the percentage of each subset. HCQ, Hydroxychloroquine; AV, annexin V; PI, propidium iodide.
Inhibition of Bcl-xL, but not Bcl-2, promoted apoptosis in HCQ-treated BxPC-3 cells. ABT-263 inhibits Bcl-2, Bcl-xL, and Bcl-w, and ABT-199 inhibits Bcl-2 (7, 8, 19). Therefore, the expression of anti-apoptotic Bcl-2 family proteins, including Bcl-2 and Bcl-xL, was examined in the three cell lines. The three pancreatic cancer cell lines were positive for Bcl-xL. Bcl-xL expression was greater in BxPC-3 cells than in the other two cell lines (Figure 4A). Bcl-2 expression in PANC-1 cells was faint compared to the other two cell lines. Next, the expression of Bcl-2 and Bcl-xL proteins in BxPC-3 cells were examined after treatment with ABT-199, the Bcl-xL inhibitor A1331852, or ABT-263 (Figure 4B). A1331852 and ABT-263 did not decrease Bcl-2 and Bcl-xL expression, but rather tended to increase them. The effects of Bcl-xL inhibition on apoptosis of HCQ-treated BxPC-3 cells were also examined. The combination of HCQ with A1331852 or ABT-263 significantly increased the percentage of early apoptotic BxPC-3 cells more profoundly compared with HCQ and ABT-199 (Figure 4C). Unlike ABT-263, A1331852 significantly promoted late apoptosis of HCQ-treated BxPC-3 cells. Representative results are shown in Figure 4D. These results suggest that ABT-263 promoted HCQ-induced apoptosis via Bcl-xL inhibition.
Bcl-xL inhibition promotes apoptosis in HCQ-treated BxPC-3 cells. (A) The protein levels of Bcl-2 and Bcl-xL were examined by immunoblotting. β-actin was used as a loading control. The band densities were measured using the ImageJ and normalized to those of β-actin. (B) BxPC-3 cells were cultured with HCQ (10 μM) and ABT-199 (2.5 μM), A1331852 (0.625 μM), or ABT-263 (2.5 μM) for 2 days. Similarly, the protein levels of Bcl-2 and Bcl-xL were examined. (C) BxPC-3 cells were cultured with HCQ (10 μM) with or without ABT-199 (2.5 μM), A1331852 (0.625 μM), or ABT-263 (2.5 μM) for 2 days. The harvested cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. The data are means±SD, analyzed using Student’s t-test (*p<0.05, **p<0.01, N.S.: not significant). The experiments were performed in triplicate and repeated twice. (D) Representative results are shown; numbers represent the percentage of each subset.
We further confirmed these results using siRNA-mediated knockdown of Bcl-xL or Bcl-2. Transfection with Bcl-2 siRNA and Bcl-xL siRNA decreased the protein expression of Bcl-2 and Bcl-xL, respectively, in BxPC-3 cells (Figure 5A). In addition, although siRNA-mediated knockdown of Bcl-2 increased the percentage of late apoptotic BxPC-3 cells, higher levels of apoptotic cells were observed in Bcl-xL-siRNA-transfected cells (Figure 5B). Representative results are shown in Figure 5C.
siRNA-mediated Bcl-xL inhibition promotes apoptosis in HCQ-treated BxPC-3 cells. (A) BxPC-3 cells were transfected with the indicated siRNAs. Three days later, the cells were harvested, and Bcl-2 and Bcl-xL protein expression was examined by immunoblotting. β-actin was used as the loading control. The band density was measured using ImageJ and normalized to β-actin. (B) BxPC-3 cells, transfected with the indicated siRNAs 2 days previously, were cultured with or without HCQ (10 μM) for 2 days. The harvested cells were stained with annexin V-FITC and PI and analyzed by flow cytometry. The data are means±SD, analyzed using Student’s t-test (**p<0.01, N.S.: not significant). The experiments were performed in triplicate and repeated twice. (C) Representative results are shown; the numbers represent the percentages of each subset. HCQ: Hydroxychloroquine; AV: annexin V; PI: propidium iodide.
In vivo antitumor effect of HCQ and ABT-737 combination on BxPC-3 cells. We finally examined the in vivo effect of HCQ and ABT-737, an analogue of ABT-263 in a xenograft mouse model. Given that ABT-263 are orally administered (7, 8) and that oral administration of ABT-263 failed to show any antitumor effects on tumor-bearing nude mice in our previous study (10), we used ABT 737 because its specificity is identical to ABT-263 but it can be administered systemically (10, 11). The experimental protocol is summarized in Figure 6A. When the treatment was started, the tumor diameter, volume and body weight were 5-6 mm, 62.5-108 mm3 and 19.6-21.4 g, respectively. The administered doses of HCQ (60 mg/kg) and ABT-737 (50 mg/kg) were determined based on previous reports (10, 11, 20). In the xenograft model, either HCQ or ABT-737 alone showed no significant antitumor effect, but combination of them slightly but significantly suppressed the growth of BxPC3 on days 18 and 21 after starting the treatment compared with the untreated group (p<0.05) (Figure 6B and C). However, this significant effect disappeared on day 25 because variations of tumor size increased. On the other hand, the combination treatment significantly decreased the body weight on days 7 and 14 after starting the treatment (p<0.01) (Figure 6D and E). This body weight loss appeared to depend on effects of ABT-737 because ABT-737 alone similarly decreased the body weight. However, the body weight loss was recovered on day 25; i.e., 11 days after the final treatment. On day 25 after starting the treatment, all mice were alive and euthanized; the tumor diameter, volume and body weight were 7.5-10 mm, 255-550 mm3 and 19,1-24.3 g, respectively.
In vivo antitumor effects of HCQ and ABT-737 on the growth of BxPC-3 cells. (A) The experimental protocol is shown. When the tumor diameter reached around 5-6 mm, the mice were divided into four groups. Each group consisted of 6 mice. HCQ (60 mg/kg) was intraperitoneally administered on days 0, 1, 3, 4, 6, 7, 9, 10, 12, and 13 after grouping (orange arrowheads). On days 2, 5, 8, 11, and 14 after grouping, cancer-bearing mice were administered intraperitoneally ABT-737 (50 mg/kg) (black arrows). The tumor size and body weights were monitored twice weekly. (B) Each line represents the tumor volume of each individual mouse. (C) The tumor volumes on days 18, 21, and 25 are shown. (D) The mean values of body weights of six mice are shown. (C) The body weights on days 0, 7, 14, and 25 are shown. Saline and DMSO were administered as vehicle controls for the HCQ and ABT-737 treatments, respectively, in the same volumes. The data are the mean values±SEM of six mice and experiments were performed twice. *p<0.05, **p<0.01 (ANOVA with Tukey–Kramer test). HCQ, Hydroxychloroquine.
Discussion
Pancreatic cancer is highly resistant to treatment and has a poor prognosis (17, 18). Although numerous efforts have been made to develop effective therapies, they have been unsuccessful. Inhibition of autophagy has received significant attention as an adjuvant therapy for pancreatic cancer patients (21). Inhibition of autophagy attenuates pancreatic cancer growth independent of TP53 status (22) and increases the sensitivity of human pancreatic cancer cells to chemotherapeutic drugs (23, 24). Although CQ is more toxic than HCQ, we previously reported that it can sensitize human pancreatic cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (25). In this regard, new CQ analogues have been developed, and these can exert antitumor effects on BxPC-3 cells (26). In addition, the less toxic HCQ can enhance PI3K/mTOR inhibitor-induced antitumor effects in pancreatic cancer cells (27). Furthermore, HCQ has been administered clinically to pancreatic cancer patients (14-16). Based on these lines of evidence, the effects of HCQ on human pancreatic cancer cells in combination with inhibitors against anti-apoptotic Bcl-2 family proteins were investigated in the present study. Interestingly, it has been reported that designed nanocarriers containing HCQ and CDK4/6 inhibitor can be a promising treatment for pancreatic cancer, especially when combined with ABT-737 (28).
The dead BxPC-3 cells were positively stained with annexin V (Figure 3A and B). In addition, assays with caspase inhibitors revealed that the cell death induced by both HCQ and ABT-263 was completely inhibited by a pan-caspase inhibitor, indicating caspase-dependent apoptosis (Figure 3C and D). Furthermore, the cell death was partially inhibited by either a caspase-8 inhibitor or a caspase-9 inhibitor. In general, caspase-8 and caspase-9 are responsible for extrinsic and intrinsic (mitochondrial) apoptosis pathways, respectively (29), and the activation of caspase-8 triggers the cleavage of caspase-9 via truncation on Bid (30). What type of extrinsic signals that activates caspase-8 is currently unknown. Additional studies are needed to elucidate the precise mechanism.
How HCQ promotes ABT-263-induced apoptosis in BxPC-3 cells is unclear. The most plausible mechanism is that HCQ inhibited autophagy in BxPC-3 cells. HCQ inhibits lysosomal acidification and blocks autophagy by influencing autophagosome fusion and degradation (31). Autophagy is a complex cellular mechanism that maintains cell homeostasis under stress. Autophagy promotes tumor development by helping the maintenance of genomic stability. Autophagy degrades and recycles cellular components to meet the metabolic needs for growth and renders cancer cells therapy-resistant (32). Autophagy is a self-protective response of living cells or organisms to various stressful conditions, and the inhibition of autophagy causes activation of the apoptosis machinery (33-35). Therefore, in many reports, the inhibition of autophagy was suggested to restore the susceptibility of cancer cells to anticancer therapies (36, 37).
Although HCQ alone exerted antitumor effects in the three human pancreatic cancer cell lines, HCQ promoted ABT-263-induced apoptosis only in BxPC-3 cells. In addition, a similar combination effect was observed when HCQ was treated with the Bcl-xL inhibitor A1331852, but not with the Bcl-2 inhibitor AB-199 (Figure 4). This was confirmed by siRNA-mediated Bcl-xL knockdown experiments (Figure 5). We examined the expression of Bcl-2 and Bcl-xL in the three cell lines and found that Bcl-xL expression was greater in BxPC-3 cells than in the other two cell lines (Figure 4A). Given that treatment with these inhibitors did not decrease Bcl-2 or Bcl-xL expression in BxPC-3 cells (Figure 4B), these inhibitors appeared to exert their effects via functional Bcl-xL inhibition. However, why these combination effects were observed only in BxPC-3 cells remains unclear. Our hypothesis is that BxPC-3 cells are sensitive to combination treatment because this cell line expresses a higher Bcl-xL level compared with the other two cell lines (Figure 4A), and that therapy resistance is mainly dependent on Bcl-xL. However, given that HCQ can induce apoptosis in chronic lymphocytic leukemia via down-regulated Bcl-2 expression (38), Bcl-2 and Bcl-xL could play protective roles against apoptosis in hematological malignancies and solid tumors, respectively.
We used ABT-737 in the xenograft model because it has the same specificity and can be administered systemically in vivo (10, 11). Although the combination effect of HCQ and ABT-737 on BxPC3 growth was slight, the difference compared with the untreated group was significant (Figure 6B and C). On the other hand, the combination therapy transiently but significantly induced body weight loss (Figure 6D and E), but the body weight loss was recovered. Although Bcl-xL inhibition induces thrombocytopenia (7), we did not observe any bleeding in ABT-737-treated mice. Currently, the cause of the body weight loss is unknown, this weight change could be a result of drug toxicity and this needs to be investigated in future work.
Unexpectedly, ABT-199 significantly increased PANC-1 cell viability when cultured with or without HCQ (10 μM) (Figure 2A). Given that ABT-263 failed to show such an effect, it appeared to be dependent on Bcl-2 inhibition. Interestingly, Bcl-2 expression in PANC-1 cells was lower than that in the other two cell lines (Figure 4A), suggesting that roles of Bcl-2 in PANC-1 cells might be not so crucial. The mechanism is currently unknown but could involve off-target effects. Further analysis is required to elucidate the underlying mechanisms.
Conclusion
We showed that HCQ effectively promoted ABT-263-induced apoptosis in BxPC-3 human pancreatic cancer cells, and that ABT-263 in a combined treatment was suggested to be due to Bcl-xL inhibition. These findings indicate that HCQ is a promising modality to augment the therapeutic efficacy of ABT-263 against pancreatic cancer cells. However, the effect was observed only in BxPC-3 cells among three cell lines. The observed findings have to be confirmed by using freshly-isolated human pancreatic cancer cells after surgery.
Acknowledgements
This study was supported by JSPS KAKENHI Grant (no. 21K07177 to M. Harada) and by the Shimane University “SUIGANN” Project. The work was supported by JST SPRING Grant Number JPMJSP2155.
Footnotes
Authors’ Contributions
MMH, YI, and MH designed the research, performed experiments, and wrote the paper. HK, IDK, and MH analyzed data.
Conflicts of Interest
The Authors declare no competing interests concerning this study.
- Received March 21, 2022.
- Revision received May 21, 2022.
- Accepted May 23, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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