| BACKGROUND AND OBJECTIVE Of all cancers reported in women, breast cancer presents with the highest incidence and the second highest mortality. The current fundamental treatment for breast cancer is radiation therapy (RT) which is widely used and effective. Nevertheless, tumor cells in hypoxic regions usually exhibit remarkable radio-resistance, which undoubtedly poses great challenges for successful RT treatment. Besides, hypoxic regions are found present in most solid tumors, but seldom form in normal tissues. Therefore, tumor hypoxia offers an attractive tumor-specific target for improving the response to ionizing radiation.As a prototypic tumour suppressor gene, p53 plays an important role in the regulation of radiosensitivity, lack of p53 can lead to resistance toward radiation treatment in different malignant tumors. It has been reported that the wild-type p53 tumor cells was more sensitive to irradiation than p53 knockout cell line in hypoxia, hypoxia may provide an environment that selects for cells losing of p53 function. Moreover, hypoxia may decrease tumor cell radiosensitivity through the suppression of p53 activity in some tumor cell lines, the reactivation of p53 proves to be an effective way of targeting hypoxic tumors. Since the hypoxic regions are seldom form in normal tissues, synthesized p53 peptides specifically target hypoxic breast cancer cells can be a novel approach to radiosensitization.Mitophagy, a type of autophagy in mitochondria, plays a critical role in the selective removal of damaged or unwanted mitochondria to avoid cell death or trigger the alternative cell-death pathway. As a central factor in mitophagy, the protein Parkin is selectively recruited to dysfunctional mitochondria with low membrane potential, and could subsequently mediate the degradation of impaired mitochondria by autophagosomes. Meanwhile, it has been found that Parkin-mediated mitophagy could also be induced by hypoxia, which could then restore cell homeostasis by degrading defective mitochondria. Since autophagy has been reported to be implicated in the tumor resistance to radiotherapy. So we assume that Parkin-mediated mitophagy may contribute to hypoxia-induced radio-resistance.While p53 can regulate the autophagy as a transcription factor in the nucleus, it also possesses an extranuclear function to inhibit the process of autophagy by a poorly known mechanism. Moreover, p53 is also consolidated by increasing evidences able to inhibit mitophagy in normal cells. Consistently, cytosolic p53 can also disturb the process of mitophagy by an inhibitory interaction with Parkin in mouse heart cells and pancreatic β-cells. Despite the recent studies on how p53 regulate mitophagy in normal cells, the role of p53 in mitophagy in tumor cells remains poorly understood.Our previous studies successfully constructed a novel fusion protein consisting of wild-type p53 and TAT domain47-57 and the minimum oxygen-dependent degradation domain 557-574 (ODD) of HIF-la. TAT-ODD-p53 (TOP) was successfully delivered into cells via the TAT protein transduction domain and selectively stabilized in hypoxic tumor tissues under the regulation of the ODD domain. In this study, we evaluated the targeted radio-sensitization of TAT-ODD-p53 in vitro and in vivo and elucidated the potential underlying functional mechanisms. Our results suggested that TAT-ODD-p53 has significant radio-sensitizing activities against hypoxic breast tumor cells and these effects are mediated by inhibiting Parkin-mediated mitophagy.METHODS1. Transmembrane delivery, location and stability of synthesized p53 peptides in vitro and in vivo1.1 Cell lines and cultureMCF-7 cells and MDA-MB-157 cells were maintained in DMEM supplemented with 10% FBS. All of the cells were cultured at 37℃ under 5%CO2 in air.1.2 Peptide synthesisTAT-ODD-p53, TAT-p53, TAT-ODD-EGFP and p53 expression plasmids were constructed. Protein expression and purification were carried out as previously described. All of the proteins were stored in 50 mM PBS at-80℃ and used within six months.1.3 HypoxiaHypoxia was achieved in a Forma 1029 anaerobic chamber with humidified atmosphere of 0.5% O2,5% CO2 balanced with N2 at 37℃. The cells were cultured under hypoxic conditions for 8 h before either being treated with synthesized p53 peptides or irradiated.1.4 Western blot analysisWhole cell lysates were harvested and western blot analysis was conducted as previously described. Mitochondrial and cytosolic fractions were isolated using a QproteomeTM Mitochondria Isolation kit according to the manufacturer’s instructions. Primary antibodies included rabbit anti-cleaved caspase-3, anti-p53, anti-Parkin, anti-ATG7 anti-COX IV and anti-β-actin. All of the western blots were visualized using an ECL detection kit.1.5 Tumor modelFor the in vivo experiments,1 x 107 MDA-MB-157 cells were subcutaneously injected into the breast of 6 to 8-week-old female Balb/c immunodeficient mice as previously described. For the assessment of p53 fusion protein distribution and stability in the tumor tissues, p53 fusion proteins (1 mg/kg) or PBS were injected i.p. into adult mice with established tumors of approximately 200 mm in size for five days (n= 3 per group). To visualize viable hypoxic cells, animals were injected with pimonidazole (60 mg/kg I.P.) 60 min prior to killing.2. Radiosensitization of breast cancer cells by synthesized p53 peptides in vitro2.1 Cytotoxicity testThe cytotoxicity of synthesized p53 peptides on MCF-7, and MDA-MB-157 cells was studied to determine the concentrations to be used in the subsequent studies. The cells were plated in 0.1 mL medium in 96-well plates and incubated overnight. On the following day, the cells were exposed to p53 peptide dissolved in PBS (0,2,4, 8,16,32 μg/ml) for 72 h under normoxia or hypoxia.20 ml of 5 mg/mL MTT solution in PBS was added to each well for 4 h at 37℃. A 20% sodium dodecyl sulfate (SDS) solution in 0.01% HCl (150 μL) was added to each well, before the measurement under an absorbance at 570 nm using a spectrophotometric plate reader. Two independent experiments were performed, each in triplicate. The inhibitory concentrations (IC) values were determined for each cell line.2.2 Colony formation assaysThe colony formation assay was performed to determine radiosensitivity. The cells were plated in 6-well plates and allowed to adhere overnight before the exposure to radiation at indicated doses using 6-MV X-ray generated by a linear accelerator at a dose rate of 5 Gy/min. After incubation for 10-14 days, the cells were stained with 0.5% crystal violet in methanol. The colonies (a population of>50 cells) were then counted using microscopy. The survival data from different experiments were pooled, and survival curves were fitted and analyzed using the"Multitarget-single hitting" model. The sensitizing enhancement ratio (SER) and oxygen enhancement ration (OER) were calculated as previously described.2.3 Flow cytometric analysis of apoptosisFITC-conjugated annexin V was used to detect the presence of apoptosis. The cells were seeded in 6-cm dishes and grown to 80% confluence. After irradiation, the cells were incubated for 24 h and then harvested and stained with annexin V-FITC and PI according to the manufacturer’s instructions. Fluorescence was detected by flow cytometry.3. Radiosensitization of breast cancer cells by synthesized p53 peptides in vivo3.1 Tumor growth inhibition studyThe inhibition of tumor growth in mice with tumor grafts of approximately 200 mm3 was studied by injecting mice i.p. every day for a total of five days with p53 fusion proteins (1 mg/kg) or PBS (n= 5 per group). A single dose (10 Gy) was applied at day 5. The tumor volume was measured with calipers using the formula V = (a×b2)/2, in which a and b are the largest and the smallest perpendicular diameters, respectively.3.2 Tissue apoptosis assay by TUNELTerminal deoxynucleotidyl transferased UTP nick end-labeling (TUNEL) was performed on frozen tumor sections seven days post-exposure, using an In Situ Cell Death Detection Kit according to the manufacturer’s instructions. After three 5-min washes in PBS, the samples were mounted in fluorescence mounting medium with DAPI to mark the nuclei All of the matched samples were examined with a fluorescence microscope.4. Mechanism by which TAT-ODD-p53 sensitize the hypoxic breast cancer cells4.1 Preparation of stable GFP-LC3-expressing cellsA recombinant lentivirus containing GFP-LC3 was obtained from Genepharma. MDA-MB-157 and MCF7 cells were infected with lentivirus particles and isolated via fluorescence-activated cell sorting to obtain the cells that stably expressed GFP-LC3.4.2 RNA interference and PlasmidsThe p53 siRNA, Parkin siRNA, ATG7 siRNA and plasmid encoding Parkin were obtained from Genepharma. The transfections were performed using Lipofectamine 2000 reagent according to the instructions provided by the manufacturer.4.3 Immunocytochemistry and confocal microscopyA total of 5×104 cells were plated in a confocal dish overnight. The cells were cultured under hypoxic (0.5%O2) or normoxic (20%O2) conditions for 8 h before irradiation. After irradiation (6Gy), the cells were cultured for 6h before fixed for thirty minutes with 4% paraformaldehyde, the cells were loaded with 200 nM MitoTracker Red at 37℃. They were then mounted with DAPI mounting medium (Invitrogen) and observed under a fluorescence confocal microscope. Confocal imaging was carried out using the Olympus FV10-ASW software.4.4 Transmission electron microscopyA total of 5×105 cells were seeded in 6-cm dishes overnight, cultured under hypoxic (0.5%O2) conditions for 8 h. After irradiation (6Gy), the cells were cultured for 6h before harvested and kept overnight in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) before being cut into 50-μm sections on a vibratome. Selected areas were processed by postfixation in 1% osmium tetroxide for 1 h, dehydrated in graded ethanols and embedded in epoxy resin. Polymerization was performed at 80℃ for 24 h. Ultrathin sections (100 nm) were cut and stained with uranylacetate and lead citrate, and viewed under AMTTEM camera systems.4.5 mtDNA assayTotal cellular DNA was extracted with a Universal Genomic DNA Extraction Kit according to the manufacture’s protocol. Real-time-PCR was performed using the PrimeScript(?)RT reagent kit and was measured by ABI 7500 Sequence Detection System.4.6 qRT-PCRTotal RNA was extracted from the cells using TRIzol reagent according to the manufacture’s protocol. The reverse-transcription used the PrimeScript(?)RT reagent kit. Real-time PCR was performed using ABI 7500 Sequence Detection System with a SYBR(?) Premix Ex TaqTM kit.4.7 Co-ImmunoprecipitationProteins were extracted from cytoplasmic fraction using the PARIS kit according to the manufacturer’s instructions. Co-immunoprecipitation was performed using the Pierce Co-Immunoprecipitation Kit as per the manufacturer’s instructions. The lysates were applied to columns containing 10μg immobilized antibodies (p53 or Parkin) covalently linked to an amine-active resin and incubated overnight at 4℃. The co-immunoprecipitate was then eluted and analyzed by SDS-PAGE along with the input controls.5. Statistical analysis:Unless stated otherwise, all of the experiments were conducted in triplicate. The data were analyzed using Student’s t-test for statistical significance. Statistical evaluations are presented as the mean ± standard deviation, and a p-value< 0.05 was considered of statistical significance.RESULTS:1. TAT-ODD-p53 was sufficiently cell-permeable and selectively stabilized under hypoxic conditionsTo assess the cell-penetrating delivery of the synthesized p53 peptides, MDA-MB-157 cells (null p53) were treated with PBS or p53 peptides at a final concentration of 10 μg/ml for 1 h in normoxic (20% O2) or hypoxic (0.5%O2) conditions. Western blotting showed that the visible expression of p53 was only found in cells that were treated with the TAT-p53 or TAT-ODD-p53 fusion proteins, suggesting that p53 fusion proteins conjugated with TAT could be effectively delivered to cells and localized intracellularly in vitro. Importantly, p53 was stabilized under both normoxia and hypoxia when the cells were treated with TAT-p53. In contrast, when the cells were treated with TAT-ODD-p53, p53 was rapidly degraded in normoxia but was consistently stable under hypoxia.2. TAT-ODD-p53 was selectively stabilized in the hypoxic regions of the tumor tissueTo assess whether TAT-ODD-p53 could selectively accumulate in the hypoxic regions of breast tumor in vivo, we used immunofluorescence staining to analyze the co-localization of TAT-ODD-p53 and pimonidazole, a probe known to selectively stain hypoxic areas. The result showed that TAT-ODD-p53 was localized with pimonidazole in similar areas of tumor tissues, indicating that TAT-ODD-p53 was targeted to and stabilized in the hypoxic regions of the tumor tissue.3. The IC50 values or TAT-ODD-p53 against the hypoxic breast tumor cellsTo choose a suitable dosage for further study, we analyzed the inhibition of cell growth induced by p53 fusion proteins in different O2 concentration environments. Breast cancer cells were incubated with a series of concentrations of p53 fusion proteins (0-32μg/ml) for 72 h under hypoxia (0.5%O2) or normoxia (20%O2). Cell viability was assessed with the MTT assay. TAT-ODD-p53 induced a significant reduction of cell viability only in the low-oxygenated condition, which suggested that TAT-ODD-p53 could selectively inhibit tumor cell growth in hypoxia. The IC50 values for TAT-ODD-p53 against the breast tumor cells in the low-oxygen condition was calculated, which were 4.00 μg/ml and 12.85 μg/ml respectively in MDA-MB-157 (null p53) and MCF-7 (wild-type p53). Subsequent experiments were carried out using a concentration of 4 μg/ml in two aforementioned cell lines.4. TAT-ODD-p53 sensitized the hypoxic breast cancer cells to ionizing, radiationTo determine the radiosensitizing effect of TAT-ODD-p53, a colony-forming assay was performed using MCF-7 and MDA-MB-157 cells exposed to radiation after an 1-h incubation with TAT-ODD-p53 under normoxic or hypoxic conditions. The radioprotective effect of hypoxia can be expressed quantitatively using the oxygen enhancement ratio (OER). The OERs of MCF-7and MDA-MB-157 cells were 1.91 and 2.43, respectively, suggesting that hypoxia induced a significant radioresistance. The sensitizer enhancement ratio (SER) was calculated as the radiation dose needed to induce a survival fraction of 37%(DO in radiobiology) using radiation alone divided by the dose needed for TAT-ODD-p53 plus radiation to induce the same survival fraction.TAT-ODD-p53 sensitized MCF-7 and MDA-MB-157 cells to ionizing radiation, with SERs up to 1.55 and 2.24, respectively.Apoptosis was measured by FCM and western blot in MDA-MB-157 cells treated with TAT-ODD-p53 (4 μg/ml) under hypoxic conditions at 48 h after irradiation. We found that the radiation-induced apoptosis under hypoxia increased notably after exposure to TAT-ODD-p53. There was a consistent and significant increase in caspase-3 cleavage in the combination group in hypoxic conditions. Taken together, these results further supported that TAT-ODD-p53 enhanced the radiosensitivity of hypoxic breast cancer cells.5. Radiosensitization of breast cancer cells by synthesized p53 peptides in vivoThe in vivo effects of TAT-ODD-p53 in combination with local tumor irradiation were evaluated. Nude mice with subcutaneous MDA-MB-157 xenograft tumors were treated with TAT-ODD-p53 (1 mg/kg, i.p.) every day for five days before a single dose of 10Gy irradiation. The tumor growth was monitored until reaching the maximal permitted volume (600 mm3). In un-irradiated mice treated with PBS or TAT-ODD-p53, the tumors reached the maximal permitted volume at day 14 and 18, respectively. The time to reach this volume was 21 days in mice treated with radiation alone, and increased to 32 days when treated by radiation combined with TAT-ODD-p53, suggestive of a distinctly supra-additive growth delay. TAT-ODD-EGFP did not inhibit the growth nor enhance the radiosensitivity of xenografts established by MDA-MB-157 in nude mice, demonstrating that TAT-ODD domain had no antitumor activity.DNA fragmentation was detected by TUNEL (TdT-mediated dUTP nick end labeling), and DAPI was used as a nuclear marker. TAT-ODD-p53 significantly increased the number of apoptotic nuclei with fragmented DNA in tumor tissues isolated at seven days post-irradiation. Consistent with previous data, TUNEL assays showed that the number of apoptotic cells significantly increased in the combination treatment group compared with single TAT-ODD-p53 or irradiation treatment group.6. mitophagy was induced by hypoxia in breast cancer cellsMitochondrial clearance by autophagic processes, termed mitophagy, is essential for mitochondrial homeostasis. The co-localization between LC3 (GFP-LC3; green fluorescence), a marker of autophagosomes and mitochondria (MitoTracker; red fluorescence) was evaluated for evaluation of mitophagy. The quantification of co-localization showed that more mitochondria were co-localized with autophagosomes in hypoxic cells treated with or without irradiation, suggesting that mitophagy was induced in low-oxygenated condition. Additionally, electron microscopy found that mitochondria were incorporated into autophagic vacuoles in hypoxic cells, further consolidating that mitophagy was induced by hypoxia in breast cancer cells.Besides, the expression of mt-Atp6, mitochondrial DNA (mtDNA) and Rp113, genomic DNA, was assessed by real time PCR, whose ratio reflected the relative number of mitochondria in each cell.The results revealed that mtDNA content decreased in irradiated cells exposed to hypoxia, which could be reversed by Atg7 knockdown,and suggested that mitophagy in the hypoxic condition participated in mitochondrial clearance.7. TAT-ODD-p53 might boost the sensitivity of hypoxic cells to irradiation through the inhibition of mitophagyImmunofluorescence of GFP-LC3 transfected MDA-MB-157 cells demonstrated that co-localization between LC3 and mitochondria was decreased, and the number of mitochondria was increased in TOP group. Mitophagy was also enhanced in hypoxic irradiated MCF7 cells (wild-type p53) transfected with siRNA for p53, and the number of mitochondria was decreased after p53 knockdown. These results suggested that TAT-ODD-p53 could inhibit hypoxia-induced mitophagy in breast cancer cells.To further clarify whether mitophagy was involved in the radio-sensitization function of TAT-ODD-p53 in hypoxia, we evaluated the radio-sensitivity of hypoxic MDA-MB-157 cells treated with TAT-ODD-p53 or/and Atg7 siRNA by colony-forming assay. The hypoxic cells were more sensitive to irradiation by inhibiting the mitophagy via Atg7 knockdown, with SERs 1.93. The radio-sensitizing effect of TAT-ODD-p53 was attenuated after autophagy process blocked by Atg7 knockdown, since the SERs of TAT-ODD-p53 in hypoxic cells transfected with Atg7(1.27) was less than control group(2.26), suggesting TAT-ODD-p53 might boost the sensitivity of hypoxic cells to irradiation, at least in part, through the inhibition of mitophagy.8. TAT-ODD-p53 was able to block mitophagy via Parkin pathway in hypoxic conditionsParkin is an E3 ubiquitin ligase, which selectively translocates onto impaired mitochondria to initiate mitophagy. Hypoxia-induced mitophagy could be inhibited via Parkin knockdown in irradiated MDA-MB-157 cells. The elevated mtDNA content in hypoxic cells depleted Parkin, suggesting the clearance of mitochondria by mitophagy was reduced. These results indicated that mitophagy might occur through the Parkin pathway in hypoxic condition.Overexpression of Parkin partly reversed the inhibitory effect of TAT-ODD-p53 on mitophagy in MDA-MB-157 cells, and the number of mitochondria also recovered in the combination group compared with TOP group. In addition, the p53 knockdown-mediated induction of co-localization between LC3 and mitochondria in hypoxia was eliminated by Parkin deletion in MCF7 cells, and the number of mitochondria was no longer decreased by p53 knockdown after Parkin was inhibited.These results demonstrated that TAT-ODD-p53 was able to block mitophagy via Parkin pathway in hypoxic conditions.9. TAT-ODD-p53 may interact with Parkin in cytoplasm to disturb its translocation to mitochondriaTo further confirm the mechanism of p53’s effect on Parkin-mediated mitophagy, the expression of Parkin was tested by qRT-PCR and western blot. P53 did not alter the expression level of Parkin visibly but affect the translocation of Parkin to mitochondria in hypoxic breast cancer cells. In MDA-MB-157 cells, Parkin translocation to mitochondria was inhibited by TAT-ODD-p53. In addition, such translocation was increased by p53 knockdown in MCF7 cells, TAT-ODD itself cannot regulate the translocation of Parkin. Since transcription-independent functions of cytoplasmic p53 have been commonly reported, and Parkin selectively translocates to impaired mitochondria to initiate mitophagy, the protein-protein interaction between p53 and Parkin was examined in cytosolic lysate of MDA-MB-157 cells. After being treated with TAT-ODD-p53, the Parkin-p53 complex was observed in both immunoprecipitates of Parkin and p53 in the cytosolic lysate under hypoxic conditions.The protein-protein interaction between endogenous p53 and Parkin was also found in hypoxic MCF7 cells. P53 may interact with Parkin in cytoplasm to disturb its translocation to mitochondria, and thereby block mitophagy process.10. TAT-ODD-p53 regulate tumor cell radiosensitivity through Parkin-mediated mitophagyRe-expression of Parkin in TAT-ODD-p53 treated MDA-MB-157 cells rescued radio-resistance in hypoxia. Moreover, inhibition of mitophagy by ATG7 knockdown reversed Parkin-induced radioresistance in TAT-ODD-p53 treated MDA-MB-157 cells in hypoxia. These data suggested that TAT-ODD-p53 could regulate tumor cell radiosensitivity through, at least in part, Parkin-mediated mitophagy.CONCLUSIONS1. TAT-ODD-p53 was sufficiently cell-permeable and selectively stabilized under hypoxic breast cancer cells both in vitro and in vivo.2. significantly enhanced the radio-sensitivity of hypoxic breast cancer cells both in vitro and in vivo.3. TAT-ODD-p53 might boost the sensitivity of hypoxic cells to irradiation, at least in part, through the inhibition of mitophagy.4. TAT-ODD-p53 inhibits mitophagy through suppressing the translocation of Parkin to mitochondria in hypoxia.5. TAT-ODD-p53 could regulate tumor cell radiosensitivity through, at least in part, Parkin-mediated mitophagy. |
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