| 1. IntroductionHypopharyngeal squamous cell carcinoma (HSCC) accounts for approximately 3 to 5% of all head and neck cancers. The prognosis of HSCC is very poor, and the 5-year overall survival rate is approximately 15 to 45%. Cisplatin (DDP)-based chemotherapy is an important part of the multimodality treatment for head and neck cancers. However, intrinsic and acquired resistance to DDP is common in HSCC treatment, and the effectiveness of chemotherapy is often severely compromised. Over the decades, it has remained difficult to effectively overcome DDP resistance in chemotherapy for head and neck cancers.Chloroquine (CQ) is widely used as an anti-malarial and anti-rheumatoid drug. Recently, CQ has been reported to enhance the efficacy of drugs and radiation in antitumor studies, including studies on prostate cancer, malignant peripheral nerve sheath tumor, hepatocellular cancer, colon cancer, breast cancer and esophageal cancer CQ significantly suppressed the growth of pancreatic cancer in vitro and in vivo as a mono-drug therapy. In contrast, CQ did not sensitize 4T1 tumors or small cell lung cancers. The CQ-induced enhancement of the antitumor effect seems to depend on the tumor type and context. It is unclear whether CQ could enhance the efficacy of DDP in treating HSCC.The CQ-mediated enhancement of antitumor efficacy has mainly been attributed to its autophagy inhibition mechanism. Autophagy is a cellular homeostatic process in which cytoplasmic components are sequestered by double-membrane structures and then transported to lysosomes for degradation and recycling. During the process of autophagy, the formation of an autophagosome (a double-membrane cytosolic vacuole that characterizes autophagy) is associated with conversion of the cytosolic-type microtubule-associated protein light chain 3 (LC3) to the membrane-bound type LC3-II. The level of LC3-II is correlated with the extent of autophagosome formation. The adaptor protein p62 sequestosome 1 (p62) can bind directly to LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. The accumulation of p62 is associated with blocked autophagy. CQ inhibits autophagy because it can affect lysosome acidification. In studies of autophagy, CQ and its analogs are often used to inhibit the degradation of LC3-II and p62 proteins to measure the autophagic flux. These are the only autophagy inhibitors that can be used clinically.The role of autophagy in cancer is complex and paradoxical. Autophagy defects can lead to increased tumorigenesis, whereas autophagy itself can promote the survival of cancer cells under stressed conditions and even facilitate tumor metastasis. The levels of Beclin-1 (a key autophagy regulator) and LC3 were downregulated in human HSCC, indicating an altered autophagy level in hypopharyngeal cancer cells. In the present study, we combined DDP and CQ, an autophagy inhibitor, as an anticancer therapy in vitro and in a xenograft mouse model with the goal of improving the treatment of human HSCC.2. Objectives(1) To examine the autophagy-modulating effects of CQ on growth of hypopharyngeal FaDu cells;(2) To clarify whether chloroquine could enhance the efficacy of DDP on hypopharyngeal FaDu cells in vitro and in the treatment of hypopharyngeal xenograft tumors and to further elucidate the underlying mechanism.3. Experiment materials and methods3.1 Cell CultureThe human hypopharyngeal FaDu cell line was obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37℃ and humidified 5% CO2.3.2 Reagents and antibodiesCisplatin, chloroquine, rabbit antibodies against LC3, Beclin-1, Bax, Bcl-2, p62, P-actin were used in the experiment.3.3 Cell viability assayCell Counting Kit (CCK-8) WST-8 was used for Cell viability assay. Formula for Cell viability calculation:cell viability%= [A (drug)-A (blank)]/[A (control)-A (blank)] x 100.3.4 RNA interferenceBeclin-1 siRNAs and scrambled siRNAs, the lentiviral shRNA to Beclin-1 were synthesized by Genepharma (Shanghai, China). Beclin-1 siRNAs (5’to 3’): GTGGAATGGAATGAGATTA; Scrambled siRNAs (5’to 3’): TTCTCCGAACGTGTCACGT.3.5 Animal modelHuman hypopharyngeal FaDu cells were used as a xenograft model in male BALB/c nude mice (5 to 6 weeks old). A suspension of 3 x 106 cells in 100 uL volume was inoculated subcutaneously into the right flank of mice. The tumor sizes averaged approximately 5×5 mm after 7 days; then, the mice were divided into 4 groups that were matched for tumor volume and treatment was initiated (7 mice per treatment group).Treatment groups consisted of vehicle control (normal saline); CQ; DDP and DDP+CQ. CQ was administered at doses of 60 mg/kg for 18 consecutive days. DDP was administered at doses of 5 mg/kg every 6 days. Three injections of DDP were given in total. In the DDP+CQ group, DDP was given 20 min after CQ administration All the drugs were given by intraperitoneal injection. After 18 days of treatment, mice were sacrificed and tumor tissues were harvested for study. A caliper was used to measure the tumors every 3 days. The tumor volume was calculated using the following formula:volume= (length×width2)/2. The body weight of mice was measured every 3 days to evaluate the systemic toxicity of the drugs. For survival analysis, mice in the CQ group and DDP+CQ group continued to receive administration of CQ at doses of 60mg/kg until they meet the death criteria. Mice were euthanized and considered dead when 1) a tumor exceeded 2 cm in the maximal dimension,2) a tumor began to cause skin ulceration and 3) a tumor caused the mouse to become moribund. The conditions of the mice were closely monitored (at least 4 times per day). Mice were sacrificed by anesthetizing with an intraperitoneal injection of 0.8% pentobarbital sodium (60 mg/kg), followed by cervical dislocation. All efforts were made to reduce pain experienced by the mice. For Beclin-1 siRNA studies, FaDu cells were infected with lentiviral siRNA to Beclin-1 or a scrambled control and subjected to a short puromycin selection; then,3 million tumor cells were injected into the flanks of nude mice. Seven days after the inoculation, mice were divided into 4 groups (n=7) matched for the tumor volume:control shRNA group, Beclin-1 shRNA group, DDP+control shRNA group, DDP+Beclin-1 shRNA group. DDP was given at doses of 5 mg/kg every 6 days and 3 injections were used in total. After treatment for 18 days, tumor tissues were collected for analysis. Measurements were performed as described above.3.6 Western blotTumor cells or tissues were collected and proteins were extracted. LC3, p62, Bax, bcl-2 were analyzed by western blots.3.7 Immunohistochemistry (IHC)LC3 and p62 protiens were determined by IHC. Tumor sections were observed under a Leica light microscope. Positive areas were quantified by Image-Pro Plus version 6.0.3.8 TdT-mediated dUTP nick end labeling (TUNEL)TUNELwas performed with an ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit to detect apoptosis.3.9 Statistical analysisComparisons were made with one-way analysis of variance or t-test. Kaplan-Meier curves for the survival of mice were analyzed with the log-rank test. Results were presented as the mean±SEM. A P value<0.05 was considered statistically significant.4. Results4.1 In vitro experiments4.1.1 Dose- and time-dependent effects of DDP on growth of FaDu cellsFaDu cells cultured in normal medium were treated with concentrations of DDP (0, 0.2,0.5,1,2.5,5μM) for 24 or 48 hours. Cell viability was analyzed with the CCK8 assay. Growth of FaDu cells was inhibited by DDP in a Dose- and time-dependent manner. The IC50 of DDP treatment for 48h was 2.512μM(2.230-2.828μM,95% CI), so we chose DDP concentration 2.5μM for the following experiments.4.1.2 Effects of CQ on growth of FaDu cellsFaDu cells cultured in normal medium were treated with concentrations of CQ (0,5, 10,15,20,25μM) for 24,48,72,96 or 120 hours. Cell viability was analyzed with a CCK8 assay. Growth of FaDu cells was not affected by the lower concentrations of CQ treatment (5 μM,10 μ) for 24,48,72,96 or 120 hours, whereas CQ treatment (15,20,25 μM) could inhibit growth of FaDu cells in a dose- and time-dependent manner.4.1.3 Autophagy inhibition by CQ in FaDu cellsFaDu cells cultured in normal medium were treated with concentrations of CQ (0, 10μM) for 24 hours. Cells were harvested with cell proteins extracted. LC3 and p62 were used as autophagic markers for Western blot analysis. LC3 and p62 protein levels in FaDu cells treated with CQ (10 μM) were markedly up-regulated, indicating ·blockade of autophagy. CQ (10μM) could effectively inhibit autophagy in FaDu cells whereas did not affect the cell viability, so we chose CQ (10μM) for the following experiments.4.1.4 Autophagy induction effect by DDP in FaDu cellsFaDu cells cultured in normal medium were treated with concentrations of DDP (0,2.5μM) for 48 hours. Cells were harvested with proteins extracted. LC3 and p62 were analyzed by Western blot assay. LC3 protein levels in FaDu cells treated with DDP (2.5 μM) were notably increased whereas accumulation of p62 protein was markedly decreased, indicating an autophagy induction by DDP treatment.4.1.5 Sensitization of FaDu cells to DDP treatment by CQ.FaDu cells cultured in normal medium were treated with concentrations of CQ (0, 10 μM) for 24 hours, then DDP (2.5μM) was added into the medium and cells were cultured for 48 hours. Cell viability was analyzed with a CCK8 assay 48 hours later. Growth of FaDu cells was markedly decreased by the CQ plus DDP treatment compared with the DDP alone treatment。4.1.6 Effects of CQ plus DDP on FaDu cell apoptosis.FaDu cells cultured in normal medium were treated with concentrations of CQ (0,10 μM) for 24 hours, then DDP (2.5 μM) was added into the medium and cells were cultured for 48 hours. Cells were harvested and proteins were extracted for Western blot analysis to detect expressions of apoptotic proteins Bax and Bcl-2. Levels of proapoptotic Bax were notably increased and levels of antiapoptotic Bcl-2 were markedly decreased in the CQ plus DDP group compared with control. The Bax/ Bcl-2 ratio was also significantly increased in the CQ plus DDP group, indicating a promotion effect of CQ on the DDP-induced apoptosis.4.1.7 Effects of Beclin-1 siRNA on FaDu cellsBeclin-1siRNA was used to inhibit the expression of autophagy related protein Beclin-1. FaDu cells were transfected with Beclin-1siRNA or a scrambled siRNA. DDP was added into the culture medium 24 hours after transfection. Cell viability of FaDu cells was analyzed after DDP treatment for 48 hours. Silence of Beclin-1 had minimal effects on the growth of FaDu cells; however, Silence of Beclin-1 could notably increase the DDP-induced inhibition of growth in FaDu cells, indicating autophagy inhibition may be the mechanism of the CQ-induced sensitization effect to DDP in FaDu cells.4.2 In vivo experiments4.2.1 CQ increased the efficacy of DDP, decreasing tumor growth and prolonging the survival of mice.We monitored the tumor volume and final tumor weight for each group. The mono-CQ therapy had no impact on the tumor volume or tumor weight compared with the control group. The mono-DDP group had a decreased tumor volume (657.0 ±66.2 mm3) and tumor weight (0.493±0.0496 g) compared with the control group (1405.0±117.2 mm3,0.981±0.0577 g,p<0.001 for both). The DDP+CQ treatment demonstrated a further decrease in the tumor volume (350.7±54.0 mm3) and tumor weight (0.263±0.0405 g) compared with the mono-DDP therapy (p<0.01 for both).The mono-CQ therapy had no effect on survival of the mice, and the mono-DDP therapy increased survival of the mice compared with that of control (p<0.01). The combination of CQ and DDP significantly increased survival of the mice compared with that of the mono-DDP treatment (p=0.0113 by log-rank test). DDP+CQ led to a robust 15.5-day increase in the median survival compared with that of DDP alone.The body weight of xenograft mice was also monitored to evaluate the systemic toxicity throughout the treatment period. CQ did not induce a loss of body weight compared with the control group. DDP caused a significant loss of body weight relative to the control group (p<0.001). DDP+CQ produced a notable body weight loss compared with that of the control group (p<0.001), but the loss of body weight was not significant from that of the mono-DDP therapy.4.2.2 Autophagy was induced by DDP and suppressed by CQ in the hypopharyngeal xenograft tumors.We used LC3 and p62 as markers of autophagy induction and inhibition. Expression levels of LC3 and p62 were examined by Western blot and IHC after treatment for 18 days. (1) In the mono-CQ treated group, the levels of LC3 and p62 were substantially increased compared with that of control, suggesting a blockade of the autophagy flux by CQ administration. (2) In the mono-DDP treated group, the levels of LC3 were increased whereas accumulation of p62 was decreased relative to control, which suggested that DDP, as a classical anti-tumor agent, caused autophagy induction in the tumors. (3) In the CQ+DDP treated group, a further increase of LC3 levels was observed compared with the mono-DDP treated group.To confirm the time needed for inhibition of autophagy, tumor bearing mice treated with CQ (60 mg/kg/day) were sacrificed after consecutive administrations for 1,3 and 7 days separately. Tumor tissues were sectioned for LC3 Immunohistochemistry. LC3 accumulation on day 1 and 3 was rare. A marked accumulation of LC3 was observed after CQ administration for 7 consecutive days, suggesting an autophagy inhibition effect by CQ untill day 7.4.2.3 CQ addition increased apoptosis in DDP-treated xenograft mice.We performed Western blot analysis of Bax and Bcl-2, and the Bax/Bcl-2 ratio was calculated. A TUNEL assay was also performed to evaluate apoptosis. As expected, DDP treatment caused marked apoptosis of tumor cells (p<0.05 relative to the control). CQ alone did not have a pro-apoptosis effect. However, the addition of CQ significantly increased the apoptosis of tumor cells induced by DDP compared with that of DDP alone (p<0.001).4.2.4 Suppression of Beclin-1 expression by shRNA enhanced the effect of DDP on xenograft tumor growth.We inhibited autophagy in FaDu cells with shRNA to Beclin-1 and assessed the effect on xenograft tumor growth. Similar to the results of CQ treatment, suppression of Beclin-1 expression by shRNA did not impact tumor growth. However, the combination of Beclin-1 suppression and DDP treatment significantly decreased tumor growth compared with DDP plus scrambled control shRNA.5. Conclusions(1) Chloroquine can inhibit autophagy of hypopharyngeal FaDu cells in vitro and in vivo;(2) The DDP-induced autophagy upregulation may contribute to DDP resistance in FaDu cells;(3) Chloroquine can increase the DDP-induced apoptosis and sensitize hypopharyngeal FaDu cells to DDP treatment in vitro and in vivo;(4) Autophagy inhibition is a possible mechanism for the chloroquine-induced sensitization of hypopharyngeal FaDu cells to DDP treatment. |
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