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As a malignant head and neck cancer, nasopharyngeal carcinoma (NPC) has high morbidity. Parkin expression has been reported to be reduced in NPC tissues and its upregulation could enhance paclitaxel-resistant cell cycle arrest. This study was performed to explore the possible mechanism of Parkin related to B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3)/BNIP3-like (NIX)-mediated mitochondrial autophagy in NPC cells. Initially, after Parkin overexpression or silencing, cell viability and proliferation were evaluated by lactate dehydrogenase and colony formation assays. JC-1 staining was used to assess the mitochondrial membrane potential. In addition, the levels of cellular reactive oxygen species (ROS) and mitochondrial ROS were detected using DCFH-DA staining and mitochondrial ROS (MitoSOX) red staining. The expression of proteins was measured using Western blot. Results showed that Parkin overexpression inhibited, whereas Parkin knockdown promoted the proliferation of paclitaxel-treated NPC cells. Besides, Parkin overexpression induced, whereas Parkin knockdown inhibited mitochondrial apoptosis in paclitaxel-treated NPC cells, as evidenced by the changes of Cytochrome C (mitochondria), Cytochrome C (cytoplasm), BAK, and Bcl-2 expression. Moreover, the levels of ROS, mitochondrial membrane potential, and LC3II/LC3I in paclitaxel-treated C666-1 cells were hugely elevated by Parkin overexpression and were all declined by Parkin knockdown in CNE-3 cells. Furthermore, Parkin upregulation activated, whereas Parkin downregulation inactivated BNIP3/NIX signaling. Further, BNIP3 silencing or overexpression reversed the impacts of Parkin upregulation or downregulation on the proliferation and mitochondrial apoptosis of paclitaxel-treated NPC cells. Particularly, Mdivi-1 (mitophagy inhibitor) or rapamycin (an activator of autophagy) exerted the same effects on NPC cells as BNIP3 silencing or overexpression, respectively. Collectively, Parkin overexpression activated BNIP3/NIX-mediated mitochondrial autophagy to enhance sensitivity to paclitaxel in NPC.
Keywords: BNIP3/NIX, mitochondrial autophagy, nasopharyngeal carcinoma, paclitaxel, Parkin
How to cite this URL:
Ni H, Liu R, Zhou Z, Jiang B, Wang B. Parkin enhances sensitivity of paclitaxel to nasopharyngeal carcinoma by activating BNIP3/NIX-mediated mitochondrial autophagy. Chin J Physiol [Epub ahead of print] [cited 2023 Nov 30]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=389961 |
Haifeng Ni, Renhui Liu, Zhen Zhou: Contributed equally
| Introduction | |  |
As an epithelial malignant tumor, nasopharyngeal carcinoma (NPC), which has high mortality rate, is characterized by early extensive local infiltration and lymphatic spread and hematogenous dissemination.[1] It was estimated that about 129,079 people were diagnosed with NPC and 72,987 patients died of NPC globally in 2018.[2] The current evidence has demonstrated that risk factors for NPC include heredity, unhealthy diets, race, environment, bad habits as well as the Epstein-Barr virus.[1] Due to its high sensitivity to radiotherapy, patients suffering from NPC at early stage mainly receive radiotherapy.[3] Clinical data have evidenced that the combination of radiotherapy and adjuvant chemotherapy has greatly improved the 5-year survival rate of NPC patients.[4] Despite the fact that paclitaxel is regarded as the first choice of chemotherapy drug for NPC, the resistance to paclitaxel is frequently encountered.[5] This study was designed to explore the effective strategies to enhance cellular sensitivity to paclitaxel chemotherapy in NPC.
Parkin, which is an E3 ubiquitin ligase, can regulate multiple pathologies, such as mitophagy and metastasis.[6],[7] An increasing number of researches have testified that Parkin is involved in the development and progression of many cancers, such as hepatocellular carcinoma, nonsmall-cell lung carcinoma, and NPC is no exception.[8],[9],[10] A case of study testified that Parkin was reduced in NPC tissues, and its upregulation could enhance paclitaxel-resistant cell cycle arrest.[10] In addition, Parkin-mediated mitochondrial autophagy is associated with the drug resistance to certain cancer treatments through removing damaged mitochondria and maintaining healthy mitochondria.[11] Being a mitochondrial outer membrane protein, B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3)/BNIP3-like (NIX) is associated with the Bcl-2 homology 3-only family which can stimulate cell death and regulate mitochondrial autophagy.[12] A case of study has verified that Parkin could ubiquitinate BNIP3/NIX, which might in turn promote its recognition by autophagic receptors, thus contributing to autophagic removal of mitochondria.[13]
This study was implemented to explore the mechanism of NPC and to figure out whether Parkin-mediated mitochondrial autophagy could enhance cellular sensitivity to paclitaxel chemotherapy in NPC, which might be of profound social and economic significance.
| Materials and Methods | | |
Cell culture and treatment
NPC cell line C666-1 was provided by BeNa Culture Collection (Shanghai, China) and CNE-3 cell line was procured from BioVector NTCC (Beijing, China). All cells were incubated in Dulbecco's Modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Inc.) and 1% antibiotics at 37°C in the presence of 5% CO2. Subsequently, all cells were treated with paclitaxel. In addition, 10 μM Mdivi-1 (mitophagy inhibitor) was employed to treat C666-1 cells and 100 nM rapamycin (an activator of autophagy) was applied to treat CNE-3 cells.
Cell transfection
Short hairpin RNAs (sh-RNAs) specific to Parkin (shRNA-Parkin-1/2) and BNIP3 (shRNA-BNIP3-1/2), plasmids carrying Parkin (OV-Parkin), and BNIP3 (OV-BNIP3) as well as their corresponding negative control (sh-NC and Ov-NC) were provided by GeneChem (Shanghai, China). The transfection of above plasmids into cells was performed using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection efficacy was tested by the Western blot.
Lactate dehydrogenase assay
Lactate dehydrogenase (LDH) as a cytosolic enzyme can release into soluble cell fraction when cells were damaged and dead; therefore, LDH release was used as an assessment indicator of cell cytotoxicity. Initially, C666-1 and CNE-3 cells were inoculated into 96-well plates at a density of 1 × 104 cell/well. Afterward, the supernatant was collected, and the cytotoxicity was estimated utilizing LDH assay (A020-2-2; Nanjing Jiancheng Bioengineering Institute, China). With the employment of a microplate reader (Shenzhen Ziker Biotechnology Co., Ltd.), the absorbance was detected at 490 nm. At 650 nm, the background optical absorbance was estimated and was subtracted from primary measurements for each well. LDH concentration was calculated as the percentage of LDH release ratio (treated cells/control cells).
Isolation of mitochondria and cytoplasm in cells
The cytosolic fractions were isolated from C666-1 and CNE-3 cells using Mitochondrial Isolation Kit (C3601; Beyotime Institute of Biotechnology, Shanghai, China) for culture cells in line with the standard protocol. In brief, cells were lysed in lysis buffer, and mitochondria were separated in mitochondria buffer. The centrifugation of supernatant was conducted to pellet the mitochondria, and the resulting supernatant was stored as the cytosolic fraction. Cytochrome C release from the mitochondria into the cytoplasm is crucial to initiate the apoptotic cascade.[14] Therefore, the expression of Cytochrome C in the cytoplasm and mitochondria was measured using Western blot.
Colony formation assay
The proliferation of NPC cells was determined by colony formation assay. C666-1 and CNE-3 cells that plated into 6-well plates at a density of 1 × 103 cells/well were maintained in the plates for 14 d to form colonies. Subsequently, the cells were subjected to 100% methanol fixation for 10 min at room temperature and 0.5% crystal violet staining for 30 min at the room temperature. Finally, the colonies containing >50 cells were imaged using a light microscope (Olympus, Japan), and the number of clones was counted using ImageJ software (Version 1.8.0) (National Institutes of Health, USA).
Measurement of reactive oxygen species
The content of reactive oxygen species (ROS) in C666-1 and CNE-3 cells was determined utilizing ROS Assay Kit (S0033S; Beyotime Institute of Biotechnology, Shanghai, China) according to the recommended protocol. 5 μmol/L dichlorodihydrofluorescein diacetate (DCFH-DA) was applied for the staining of cells for 30 min at 37°C. We observed the distribution of mitochondrial ROS (MitoSOX) with a fluorescent dye, MitoSOX red, which specifically targets mitochondrial ROS. The level of MitoSOX was evaluated using MitoSOX red (M36008; Thermo Fisher Scientific, Inc.). Then, a fluorescence microscope (Olympus, Japan) was used to evaluate the ROS levels.
Mitochondrial membrane potential assay
5,5', 6, 6'-tetrachloro 1,1', 3,3'-tetramethyl benzimidazolyl carbocyanine iodide (JC-1), a cationic dye that is shown to accumulate in energized mitochondria, was used to evaluate mitochondrial membrane potential. Initially, C666-1 and CNE-3 cells were plated into 6-well plates. The harvested cells were subjected to 10 μM JC-1 (C2006; Beyotime Institute of Biotechnology, Shanghai, China) staining for 30 min at 37°C away from the light.
Western blot assay
C666-1 and CNE-3 cells were lysed utilizing radioimmunoprecipitation assay lysis buffer (Shanghai Absin Biotechnology Co., Ltd.). The proteins were extracted from the mitochondria and cytoplasm with a mitochondria isolation kit (KeyGEN BioTECH, Jiangsu, China), and the protein concentration was quantified with bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). After the separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to membranes. Then, the membranes were impeded by 5% nonfat milk or 5% bovine serum albumin. The membranes were incubated with primary antibodies targeting Parkin (ab73015; 1:1000; Abcam), cytochrome C (ab133504; 1:5000; Abcam), BAK (ab32371; 1:10000; Abcam), Bcl-2 (ab182858; 1:2000; Abcam), microtubule-associated protein light chain 3 (LC3)II/LC3I (ab128025; 1:1000; Abcam), BNIP3 (ab109362; 1:1000; Abcam), NIX (ab109414; 1:1000; Abcam), GAPDH (ab9485; 1:2500; Abcam), β-actin (ab6276; 1:5000; Abcam), or COXIV (ab202554; 1:2000; Abcam) overnight at 4°C. On the next day, the membranes were exposed to horseradish peroxidase-labeled goat anti-rabbit secondary antibody (ab6759; 1:5000; Abcam) at room temperature for 2 h. Finally, the protein blots were visualized and analyzed with enhanced chemiluminescence (ECL; Yeasen Biotech) and ImageJ software (Version 1.8.0), respectively.
Statistical analysis
All the data that presented as the mean ± standard deviation were analyzed with GraphPad Prism 8.0 software (GraphPad Software, Inc., USA). For the demonstration of differences among multiple groups, one-way analysis of variance with Bonferroni's post hoc test was adopted. P < 0.05 meant that all experimental results exhibited statistical significance.
| Results | | |
Parkin overexpression inhibited while Parkin knockdown promoted the proliferation of paclitaxel-treated NPC cells
To figure out the role of Parkin in NPC cell proliferation, OV-Parkin was transfected into C666-1 cells and shRNA-Parkin was transfected into CNE-3 cells. After that, Western blot was applied to examine transfection efficacy and the results showed that relative to Ov-NC group, the expression of Parkin was greatly increased in C666-1 cells after the transfection with plasmids carrying Parkin gene [Figure 1]a. In CNE-3 cells, the expression of Parkin was conspicuously reduced following the transfection with shRNAs specific to Parkin by contrast with that in shRNA-NC group [Figure 1]b. Notably, Parkin had lower expression in shRNA-Parkin-2 group than that in shRNA-Parkin-1 group. Therefore, shRNA-Parkin-2 was adopted for follow-up experiments. Subsequently, LDH assay was employed to assess the toxic effects of paclitaxel on C666-1 cells and CNE-3 cells. As [Figure 1]c demonstrates, the increased LDH release rate caused by paclitaxel in C666-1 cells was further upregulated by Parkin overexpression in comparison with the Ov-NC + Paclitaxel group. Nevertheless, the increased LDH release rate in paclitaxel-treated CNE-3 cells was greatly declined by Parkin depletion when compared to the shRNA-Parkin + Paclitaxel group [Figure 1]d. In addition, compared with the Ov-NC + Paclitaxel group, Parkin overexpression tremendously reduced the number of colonies in paclitaxel-administrated C666-1 cells, whereas Parkin deficiency increased that in paclitaxel-administrated CNE-3 cells when compared with the shRNA-NC + paclitaxel group [Figure 1]e and [Figure 1]f. To sum up, the above results indicated that Parkin overexpression inhibited, whereas Parkin knockdown promoted the proliferation of paclitaxel-treated NPC cells. | Figure 1: Parkin overexpression inhibited, whereas Parkin knockdown promoted the proliferation of paclitaxel-treated nasopharyngeal carcinoma cells. (a) The expression of Parkin in transfected C666-1 cells was detected using Western blot. (b) The expression of Parkin in transfected CNE-3 cells was detected using Western blot. (c) The lactate dehydrogenase (LDH) release rate in transfected C666-1 cells was detected using LDH assay. (d) The LDH release rate in transfected CNE-3 cells was detected using LDH assay. (e) The proliferation of transfected C666-1 cells was detected using colony formation assay. (f) The proliferation of transfected CNE-3 cells was detected using colony formation assay. Results are the mean ± standard deviation. **P < 0.01, ***P < 0.001. All experiments were replicated for three times.
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Parkin overexpression induced while Parkin knockdown inhibited mitochondrial apoptosis in paclitaxel-treated NPC cells
With the purpose of exploring the effects of Parkin overexpression or Parkin knockdown on the mitochondrial apoptosis of NPC cells, the expression levels of Cytochrome C in the cytoplasm and mitochondria and apoptosis-related proteins were evaluated adopting Western blot. Relative to Ov-NC + Paclitaxel group, Parkin overexpression decreased the content of Cytochrome C in the mitochondria of paclitaxel-administrated C666-1 cells but elevated that in the cytoplasm [Figure 2]a. However, Parkin knockdown enhanced Cytochrome C expression in the mitochondria of Paclitaxel-administrated CNE-3 cells but diminished that in the cytoplasm in comparison with the shRNA-NC + paclitaxel group [Figure 2]b. Thereafter, Western blot was used to measure the protein expression of BAK and Bcl-2 in C666-1 cells and CNE-3 cells. Similarly, Parkin overexpression elevated BAK expression and diminished Bcl-2 expression in C666-1 cells, whereas Parkin depletion imparted opposite impacts on these two proteins, evidenced by declined BAK level and elevated Bcl-2 level in shRNA-Parkin + paclitaxel group, implying that Parkin overexpression could induce mitochondrial apoptosis [Figure 2]c and [Figure 2]d. | Figure 2: Parkin overexpression induced, whereas Parkin knockdown inhibited mitochondrial apoptosis in paclitaxel-treated nasopharyngeal carcinoma cells. (a) The expression of Cytochrome C in mitochondrial and cytoplasm of transfected C666-1 cells was detected using Western blot. (b) The expression of Cytochrome C in mitochondrial and cytoplasm of transfected CNE-3 cells was detected using Western blot. (c) The contents of BAK and B-cell lymphoma-2 (Bcl-2) in transfected C666-1 cells were detected using Western blot. (d) The contents of BAK and Bcl-2 in transfected CNE-3 cells were detected using Western blot. Results are the mean ± standard deviation. **P < 0.01 and ***P < 0.001. All experiments were replicated for 3 times.
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Parkin overexpression induced while Parkin knockdown alleviated autophagy of paclitaxel-treated NPC cells
To probe the role of Parkin overexpression or Parkin knockdown in autophagy in NPC cells, the production of ROS and mitochondrial membrane potentials in NPC cells were appraised with DCFH-DA staining and JC-1. Relative to the control group, paclitaxel administration elevated ROS level in C666-1 cells, which was then further elevated by Parkin overexpression [Figure 3]a. In comparison with shRNA-NC + paclitaxel group, Parkin depletion greatly reduced the increased ROS level in paclitaxel-administrated CNE-3 cells [Figure 3]b. In addition, the level of mitochondrial ROS in C666-1 and CNE-3 cells was assessed utilizing MitoSOX red. Parkin overexpression markedly enhanced mitochondrial ROS level in C666-1 cells with Paclitaxel administration, whereas Parkin knockdown remarkably diminished that in paclitaxel-administrated CNE-3 cells [Figure 3]c and [Figure 3]d. In contrast with Ov-NC + paclitaxel group, Parkin upregulation conspicuously reduced JC-1 aggregate formation but elevated JC-1 monomers level in Paclitaxel-administrated C666-1 cells [Figure 4]a. Besides, the decreased JC-1 aggregate formation and increased JC-1 monomers level in CNE-3 cells because of paclitaxel administration were reversed after depleting Parkin level when compared to the shRNA-NC + paclitaxel group [Figure 4]b. To conclude, the above results indicated that Parkin overexpression induced, whereas Parkin knockdown alleviated autophagy of paclitaxel-treated NPC cells. | Figure 3: Parkin overexpression elevated, whereas Parkin knockdown reduced the level of reactive oxygen species (ROS) in paclitaxel-treated nasopharyngeal carcinoma cells. (a) The level of ROS in transfected C666-1 cells was detected using DCFH-DA. (b) The level of ROS in transfected CNE-3 cells was detected using DCFH-DA. (c) The level of ROS in mitochondria of transfected C666-1 cells was detected using MitoSOX. (d) The level of ROS in mitochondria of transfected CNE-3 cells was detected using MitoSOX. ***P < 0.001. All experiments were replicated for 3 times.
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 | Figure 4: Parkin overexpression induced, whereas Parkin knockdown alleviated mitochondria autophagy of paclitaxel-treated nasopharyngeal carcinoma cells. (a) JC-1 aggregate formation in transfected C666-1 cells was detected using JC-1 assay. (b) JC-1 aggregate formation in transfected CNE-3 cells was detected using JC-1 assay. ***P < 0.001. All experiments were replicated for three times.
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Parkin overexpression promoted while Parkin knockdown inhibited autophagic pathway in paclitaxel-treated NPC cells
To investigate the effects of Parkin overexpression or Parkin knockdown on autophagic pathway in NPC cells, Western blot was employed for the estimation of mitochondrial autophagy proteins and their downstream-related proteins. Compared with Ov-NC + paclitaxel group, the contents of LC3II/LC3I in Paclitaxel-administrated C666-1 cells were enhanced after overexpressing Parkin, whereas Parkin deficiency greatly descended those in paclitaxel-administrated CNE-3 cells by contrast with the shRNA-NC + paclitaxel group [Figure 5]a and [Figure 5]b. Then, Western blot was applied to measure the expression of BNIP3 and NIX, and the results showed that Parkin overexpression dramatically improved the contents of BNIP3 and NIX relative to the Ov-NC + paclitaxel group, whereas the expression of BNIP3 and NIX in Paclitaxel-administrated CNE-3 cells were rapidly declined after the transfection with shRNA-Parkin when compared to the shRNA-NC + paclitaxel [Figure 5]c and [Figure 5]d. Collectively, Parkin overexpression promoted, whereas Parkin knockdown inhibited autophagic pathway in paclitaxel-treated NPC cells. | Figure 5: Parkin overexpression promoted, whereas Parkin knockdown inhibited autophagic pathway in paclitaxel-treated nasopharyngeal carcinoma cells. (a) The expression of LC3II/LC3I in transfected C666-1 cells was detected using Western blot. (b) The expression of LC3II/LC3I in transfected CNE-3 cells was detected using Western blot. (c) The expression of BNIP3 and NIX in transfected C666-1 cells were detected using Western blot. (d) The expression of BNIP3 and NIX in transfected CNE-3 cells were detected using Western blot. Results are the mean ± standard deviation. ***P < 0.001. All experiments were replicated for 3 times.
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Parkin-induced BNIP3/NIX-mediated mitochondrial autophagy to enhance cellular sensitivity to paclitaxel
With the aim of further discussing the mechanism of Parkin in the cellular sensitivity to paclitaxel, sh-RNAs targeting BNIP3 was transfected into C666-1 cells, and plasmids carrying BNIP3 gene were transfected into CNE-3 cells. After that, Western blot was applied to test the transfection efficacy. As [Figure 6]a depicted, the expression of BNIP3 in C666-1 cells was markedly decreased by shRNA-BNIP3 by contrast with the control group. Notably, BNIP3 had lower expression in shRNA-BNIP3-1 than that in shRNA-BNIP3-2. Therefore, shRNA-BNIP3-1 was applied for follow-up experiments. Obviously, the level of BNIP3 in CNE-3 cells was tremendously enhanced after overexpressing BNIP3 when compared to the Ov-NC group [Figure 6]b. Then, Parkin and BNIP3 expression in NPC cells after transfection with or without paclitaxel treatment was evaluated by Western blot. As shown in [Figure 6]c, BNIP3 silencing significantly downregulated BNIP3 expression but had no obvious effect on Parkin expression compared with the Ov-Parkin + Paclitaxel + shRNA-NC group. Besides, when compared with the shRNA-Parkin + Paclitaxel + Ov-NC group, BNIP3 expression was notably elevated, whereas Parkin expression had no significant change in the shRNA-Parkin + Paclitaxel + Ov-BNIP3 group [Figure 6]d. Mdivi-1, initially identified as a nonspecific inhibitor of Drp1 GTPase, not only impedes Drp1-dependent mitochondrial fission but also demonstrates moderate antioxidant activity.[15],[16] A growing body of literature using Mdivi-1 as a mitophagy inhibitor for scientific research.[8],[17],[18] To further investigate the reaction mechanism of mitochondrial autophagy, Mdivi-1 (10 μM, an inhibitor of mitophagy) and rapamycin (100 nM, an activator of autophagy) were employed to administrate C666-1 cells and CNE-3 cells, respectively. Compared with Ov-Parkin + Paclitaxel + shRNA-NC group, BNIP3 deficiency diminished LDH release rate in Ov-Parkin + Paclitaxel + shRNA-BNIP3 group [Figure 7]a. It was worth noting that Mdivi-1 exhibited suppressive impacts on LDH release rate in paclitaxel-administrated C666-1 cells. On the contrary, the decreased LDH release rate caused by Parkin knockdown in paclitaxel-administrated CNE-3 cells was elevated after overexpressing BNIP3 when compared to the shRNA-Parkin + Paclitaxel + Ov-NC group [Figure 7]b. Evidently, rapamycin enhanced LDH release rate in paclitaxel-treated CNE-3 cells. In addition, the reduced cell proliferation in Ov-Parkin + Paclitaxel + shRNA-NC group was promoted by BNIP3 deficiency. Meanwhile, Mdivi-1 also increased the proliferative ability of paclitaxel-treated C666-1 cells [Figure 7]c. The enhanced proliferation of paclitaxel-administrated CNE-3 cells with Parkin interference was slightly declined by BNIP3 overexpression relative to the shRNA-Parkin + Paclitaxel + Ov-NC group. Furthermore, rapamycin diminished the proliferative ability of paclitaxel-administrated CNE-3 cells [Figure 7]d. In C666-1 cells, BNIP3 depletion elevated Cytochrome C in mitochondria but reduced that in cytoplasm by contrast with the shRNA-Parkin + Paclitaxel + Ov-NC group. In addition, Mdivi-1 was also found to upregulate Cytochrome C expression in the mitochondria of C666-1 cells and downregulate that in cytoplasm when compared with that in paclitaxel group [Figure 8]a. Compared with shRNA-Parkin + Paclitaxel + Ov-NC group, BNIP3 overexpression reduced Cytochrome C in mitochondria of CNE-3 cells but enhanced that in cytoplasm. It was noted that rapamycin administration exhibited the same impacts as BNIP3 depletion on Cytochrome C in both mitochondria and cytoplasm [Figure 8]b. Furthermore, BNIP3 interference reduced BAK expression but elevated Bcl-2 content in contrast with the Ov-Parkin + Paclitaxel + shRNA-NC group. Similarly, Mdivi-1 also reduced BAK level and ascended Bcl-2 content in paclitaxel-treated C666-1 cells compared with those in paclitaxel group [Figure 8]c. Compared with shRNA-Parkin + Paclitaxel + Ov-NC group, BAK expression was enhanced, and Bcl-2 level was depleted after transfecting BNIP3 overexpression plasmids into CNE-3 cells [Figure 8]d. Evidently, rapamycin administration exhibited the same impacts as BNIP3 overexpression on these two proteins. | Figure 6: Parkin induced the activation of BNIP3/NIX signaling in paclitaxel-treated nasopharyngeal carcinoma cells. (a) The expression of BNIP3 in transfected C666-1 cells was detected using Western blot. (b) The expression of BNIP3 in transfected CNE-3 cells was detected using Western blot. (c) Parkin and BNIP3 expression in C666-1 cells after transfection with or without paclitaxel treatment was evaluated by Western blot. (d) Parkin and BNIP3 expression in CNE-3 cells after transfection with or without paclitaxel treatment was evaluated by Western blot. Results are the mean ± standard deviation. ***P < 0.001. All experiments were replicated for 3 times.
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 | Figure 7: Parkin-induced BNIP3/NIX-mediated mitochondrial autophagy to inhibit the proliferation of paclitaxel-treated nasopharyngeal carcinoma cells. (a) Lactate dehydrogenase (LDH) release rate in transfected C666-1 cells was detected using LDH assay. (b) LDH release rate in transfected CNE-3 cells was detected using LDH assay. (c) The proliferation of transfected C666-1 cells was detected using colony formation assay. (d) The proliferation of transfected CNE-3 cells was detected using colony formation assay. Results are the mean ± standard deviation. *P < 0.05, **P < 0.01, and ***P < 0.001. All experiments were replicated for 3 times.
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 | Figure 8: Parkin-induced BNIP3/NIX-mediated mitochondrial autophagy to promote the mitochondrial apoptosis of paclitaxel-treated nasopharyngeal carcinoma cells. (a) The expression of Cytochrome C in mitochondrial and cytoplasm of transfected C666-1 cells was detected using Western blot. (b) The expression of Cytochrome C in mitochondrial and cytoplasm of transfected CNE-3 cells was detected using Western blot. (c) The contents of BAK and B-cell lymphoma-2 (Bcl-2) in transfected C666-1 cells were detected using Western blot. (d) The contents of BAK and Bcl-2 in transfected CNE-3 cells were detected using Western blot. Results are the mean ± standard deviation. ***P < 0.001. All experiments were replicated for 3 times.
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| Discussion | | |
NPC, which is one of the most common malignancies, has hidden symptoms at early stage and is easy to infiltrate and metastasize, leading to the increased proportion of patients diagnosed at advanced stages.[4],[19] Parkin, which belongs to RBR E3 ubiquitin ligases, ubiquitinates various cytosolic proteins as well as outer mitochondrial membrane proteins upon mitochondrial depolarization.[20],[21] Accumulated researches have evidenced that Parkin was involved in the progression of plenty of cancers. Take liver cancer as an example, the activation of Parkin could enhance the activity of mitophagy and promote cancer cell survival in the presence of matrine.[22] Ding et al. held the opinion that the regulation of Parkin could mediate cell proliferation, migration, apoptosis as well as metabolism in gastric cancer.[23] More importantly, Jiang et al. have verified that Parkin was downregulated in NPC tissues and Parkin upregulation could enhance sensitivity to paclitaxel in NPC.[10] In this study, it was discovered that Parkin expression was greatly increased in Parkin-overexpressed C666-1 cells but markedly decreased in Parkin-depleted CNE-3 cells. It is reported that the leakage of LDH can be an indicator of cell injury.[24] To address the toxic effects of paclitaxel on C666-1 and CNE-3 cells, LDH assay was performed, and the results demonstrated that Parkin overexpression remarkably increased LDH release rate in paclitaxel-administrated C666-1 cells, whereas Parkin depletion exhibited suppressive impacts on that in CNE-3 cells with paclitaxel treatment. In addition, the abnormal and uncontrolled proliferation is a typical feature of cancer and the intervention of cell proliferation is believed to be an effective method for the treatment of NPC.[25],[26] In the present study, it was discovered that the proliferation of paclitaxel-administrated C666-1 cells was conspicuously diminished by Parkin overexpression while that in paclitaxel-administrated CNE-3 cells was partially enhanced after interfering Parkin expression.
As is known to all, impaired mitochondrial apoptosis pathway is a common mechanism for the cancer cells escaping from the programmed cell death.[27] Interestingly, it was evidenced that mitochondrial apoptosis was a critical player in the development of NPC and the inhibition of mitochondrial apoptosis by RCN2 facilitated the progression of NPC.[28] Besides, Mao et al. have testified that Parkin could regulate mitochondrial apoptosis in many cancers, such as breast cancer[29] and hepatocellular carcinoma,[8] which indicated the important role of Parkin in cancer development. Cytochrome C is an electron transporter of the mitochondrial respiratory chain and the release of Cytochrome C in cytoplasm may activate the intrinsic pathway of apoptosis.[30] Herein, it was discovered that Parkin overexpression decreased Cytochrome C expression in the mitochondria and increased that in the cytoplasm of paclitaxel-administrated C666-1 cells, accompanied by elevated BAK expression and reduced Bcl-2 expression, implying that Parkin overexpression could induced mitochondrial apoptosis in NPC.
Mitochondrial autophagy has been viewed as an appealing therapeutic strategy for cancers.[31] Parkin is also the most characteristic pathway involved in mitochondrial autophagy and previous study evidenced that Parkin-mediated mitochondrial autophagy was involved in the drug resistance in the therapy of hepatocellular carcinoma and breast cancer by removing damaged mitochondria and maintaining healthy mitochondria.[11] Meanwhile, PINK1/Parkin-mediated mitochondrial autophagy could regulate the sensitivity of NPC cells to radiotherapy.[32] It is believed that the production of ROS is a signal for general autophagy.[33] In this study, Parkin overexpression was testified to significantly elevate ROS level in paclitaxel-treated C666-1 cells. Moreover, the diminished JC-1 aggregate formation in C666-1 cells caused by paclitaxel administration was further reduced after overexpressing Parkin, indicating that Parkin upregulation could induce excessive mitochondria autophagy in NPC cells. In addition, Parkin overexpression could increase the expression of LC3I/LC3II, BNIP3, and NIX in paclitaxel-treated C666-1 cells, suggesting the regulatory role of Parkin in autophagic pathway in NPC.
The mitochondrial outer membrane protein BNIP3/NIX can induce cell death and mediate mitochondrial autophagy.[12] It was reported that the activation of NIX-dependent mitochondrial autophagy could suppress cancer progression.[34] Importantly, Parkin can ubiquitinate BNIP3/NIX, thus promoting its recognition by autophagy receptors.[13] In this paper, it was uncovered that BNIP3 expression was greatly declined in BNIP3-silenced C666-1 cells but conspicuously elevated in BNIP3-overexpressed CNE-3 cells. With the purposes of discussing the mechanism of BNIP3/NIX in NPC, C666-1 cells were administrated with Mdivi-1 and CNE-3 cells were treated with rapamycin. Results obtained from rescue experiments demonstrated that BNIP3 knockdown reversed the impacts of Parkin overexpression on LDH release, proliferation, Cytochrome C content, and mitochondrial apoptosis in paclitaxel-administrated C666-1 cells. Notably, Mdivi-1 exhibited the same regulatory effects as BNIP3 knockdown on these aspects in paclitaxel-administrated C666-1 cells. Collectively, the above findings revealed that Parkin induced BNIP3/NIX-mediated mitochondrial autophagy to enhance cellular sensitivity to paclitaxel in NPC.
| Conclusion | | |
This study unmasked the mechanism of Parkin in NPC and identified that Parkin overexpression activated BNIP3/NIX-mediated mitochondrial autophagy to enhance sensitivity to paclitaxel chemotherapy in NPC, which for the first time revealed a potential novel target for the enhancement of cellular sensitivity to chemotherapeutic drugs.
Data availability statement
Data will be made available on request.
Author contributions
Conceptualization, HF. Ni, RH. Liu, and Z. Zhou; methodology, HF. Ni, RH. Liu, and Z. Zhou; software, B. Jiang; validation, RH. Liu, and Z. Zhou; investigation, HF. Ni, and RH. Liu; writing – original draft preparation, HF. Ni; writing – review and editing, RH. Liu, and B. Wang; visualization, B. Wang; project administration, HF. Ni; funding acquisition, HF. Ni. All authors have read and agreed to the published version of the manuscript.
Financial support and sponsorship
This work was supported by grants from the Medical and Health Research Project of Zhejiang Province, China (Grant Numbers: 2021KY870, 2020KY698, and 2020KY695) and the Hangzhou Science and Technology Development Plan Project (Grant Numbers: 2020ZDSJ0826 and 20170533B27) and The Construction Fund of Key Medical Disciplines of Hangzhou (Grant Number: OO20200123).
Conflicts of interest
There are no conflicts of interest.
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Correspondence Address:
Haifeng Ni,
Department of Otolaryngology Head and Neck Surgery, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, No. 261 Huan Sha Road, Hangzhou, Zhejiang 310006
China
 Source of Support: None, Conflict of Interest: None DOI: 10.4103/cjop.CJOP-D-23-00076
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8] |
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