Apoptotic effect of novel pyrazolone-based derivative [Cu(PMPP-SAL)(EtOH)] on HeLa cells and its mechanism

Pyrazolone complexes have strong anti-tumor and antibacterial properties, but the anti-tumor mechanism of pyrazolone-based copper complexes has not been fully understood. In this study, the possible mechanism and the inhibitory effect of a novel pyrazolone-based derivative compound [Cu(PMPP-SAL)(EtOH)] on human cervical cancer cells (HeLa cells) was investigated. [Cu(PMPP-SAL)(EtOH)] effectively inhibited proliferation of HeLa cells in vitro with an IC50 value of 2.082 after treatment for 72 h. Cell cycle analysis showed apoptosis was induced by blocking the cell cycle in the S phase. [Cu(PMPP-SAL)(EtOH)] promoted the loss of mitochondrial membrane potential, release of cytochrome c, PARP cleavage, and activation of caspase-3/9 in HeLa cells. Additionally, [Cu(PMPP-SAL)(EtOH)] inhibited the PI3K/AKT pathway and activated the P38/MAPK, and JNK/MAPK pathways. [Cu(PMPP-SAL)(EtOH)] also inhibited the phosphorylation of Iκ-Bα in the NF-κB pathway activated by TNF-α, thus restricting the proliferation of HeLa cells which were activated by TNF-α. In conclusion, [Cu(PMPP-SAL)(EtOH)] inhibited the growth of HeLa cells and induced apoptosis possibly via the caspase-dependent mitochondria-mediated pathway. These results suggest that [Cu(PMPP-SAL)(EtOH)] can be a potential candidate for the treatment of cervical cancer.

Cervical cancer is one of the leading causes of death among women suffering from cancer worldwide, and more than 85% of the deaths due to cervical cancer occur in less developed countries of the world 1 . Since the discovery of anti-tumor property of cisplatin platinum (Pt), commercially synthesized Pt-based drugs have been playing an important role in the treatment of cervical, lung, ovarian, head, and neck cancers 2,3 . Cisplatin, oxaliplatin, and carboplatin have been approved as first-line chemotherapeutics for the treatment of carcinomas in combination with other anticancer drugs 4,5 . However, neurotoxicity, hepatotoxicity, nephrotoxicity, and drug resistance have led to reduced clinical application of Pt-based drugs 6 . Therefore, there is an urgent need for the conceptualization and preparation of new and efficacious anti-cervical cancer drugs with low toxicity.
In recent years, copper complexes have attracted much attention due to their easy synthesizability, high yield and relatively potent biological activity. Since the synthesis of a series of schiff base copper complexes in 1971, it has been reported that schiff base metal complexes have certain effects on cancer treatment 7 . A recent study found that schiff based CdCl 2 (C 14 H 21 N 3 O 2 ) complex mediated apoptosis in colon cancer cells by activating the mitochondrial pathway 8 . Polypyridyl copper complex may selectively inhibit tumor cell growth and induce apoptosis in MCF-7 breast cancer cell lines through the mitochondrial apoptotic pathway 9 . Cu(sal)(phen) effectively induced apoptosis in TNBC cells through down-regulation of anti-apoptosis proteins in these cells in vitro and in vivo 10 . A mixed-ligand copper complex [Cu(L)(phen)]·MeOH induced apoptosis in HeLa cells via accumulation of ROS 11 . Recent studies indicated that pyrazolone schiff base compounds might form novel metal complexes with metals such as platinum and copper, and these metal compounds exhibited better anti-tumor and antibacterial activities than pyrazolone 12,13 . In the initial stages of the present study, we synthesized a copper complex of the pyrazolone schiff base, called 1-phenyl-3-methyl-4-propionyl-5-pyrazolone-salicylic hydrazide-copper (II) complex [Cu(PMPP-SAL)(EtOH)]. [Cu(PMPP-SAL)(EtOH)] exhibited significant anti-tumor effects on KB cells in human carcinoma of the mouth floor and multidrug-resistant KBv200 cells, with lower acute toxicity as compared to cisplatin after intraperitoneal injection in mice 14,15 . However, the apoptosis-inducing effect of Scientific Reports | (2020) 10:18235 | https://doi.org/10.1038/s41598-020-75173-8 www.nature.com/scientificreports/ copper complexes of pyrazolone schiff base on human cervical cancer HeLa cells and its mechanism have not been fully understood.
In the present study, we investigated the growth-inhibiting activity and apoptosis-inducing effect of [Cu(PMPP-SAL)(EtOH)] on cervical cancer HeLa cells in vitro, as well as its effects on the NF-κB signaling pathway induced by TNF-α, and the MAPK cell signaling pathway.   We further preformed a colony formation assay to test the ability of cells to grow under very low density conditions. As shown in Fig. 1C,D, [Cu(PMPP-SAL)(EtOH)] inhibited colony formation of HeLa cells in a concentration-dependent manner. We found that the colony formation rate of cells in 5 μg/mL and 7.5 μg/mL compound treatment group was significantly lower than that in the control group (P < 0.01), thereby confirming that [Cu(PMPP-SAL)(EtOH)] inhibits the colony formation in HeLa cells.

Results
Release of lactate dehydrogenase (LDH) in the culture medium is an enzymatic indicator that signifies loss of membrane integrity, apoptosis, or necrosis of a cell. So an LDH assay was also performed to further validate the cytotoxic effect of [Cu(PMPP-SAL)(EtOH)] on HeLa cells. As shown in Fig. 1E, the leakage rate of LDH from HeLa cells increased in a time and concentration dependent manner after treatment with [Cu(PMPP-SAL)(EtOH)]. Compared with the control group, the release of LDH in HeLa cells was significantly increased after being treated with 7.5 μg/mL [Cu(PMPP-SAL)(EtOH)] for 24 or 48 h (P < 0.05, P < 0.01), suggesting that [Cu(PMPP-SAL)(EtOH)] can cause damage to HeLa cells. In total, these results indicate that [Cu(PMPP-SAL) (EtOH)] had a significant toxic effect on the HeLa cell.
[Cu(PMPP-SAL)(EtOH)] induced apoptosis in HeLa cells. We evaluated whether [Cu(PMPP-SAL)(EtOH)] could induce apoptosis in HeLa cells using Hoechst 33258 staining assay. After treatment with [Cu(PMPP-SAL)(EtOH)] (0, 2.5, 5, 7.5 μg/mL) for 24 h, a fewer number of cells and smaller circular morphology of the HeLa cells were observed by microscopy. As shown in Fig. 2A, cells exhibited obvious apoptotic characteristics after treatment with [Cu(PMPP-SAL)(EtOH)] for 24 h, nuclei were condensed and fragmented in the apoptotic cells. Moreover, the ultrastructural alterations were observed under transmission electron microscope. As shown in Fig. 2B, there were no typical morphological changes in control cells. But when cells were exposed   Fig. 3A, after treatment for 12 h, the proportion of the cells in the G0/G1 phase reduced from 60.49 ± 3.24% to 46.27 ± 2.42% while that of cells in the S phase was elevated from 31.84 ± 3.98% to 36.25 ± 3.21% as the action dose of the compound increased (Fig. 3B). This indicated that the cells had an S phase arrest that was dose dependent. However, this trend did not change significantly over the increase in action duration, indicating that the action was not significantly time dependent.

Effects of [Cu(PMPP-SAL)(EtOH)] on apoptosis signaling pathway in
We further preformed the expression levels assay of Iκ-Bα and P-Iκ-Bα in HeLa cells via western blot after treatment with [Cu(PMPP-SAL)(EtOH)]. As shown in Fig. 6C,E,F, phosphorylation of Iκ-Bα was inhibited as the concentration of [Cu(PMPP-SAL) (EtOH)] increased. Consequently, it can be inferred that [Cu(PMPP-SAL)(EtOH)] inhibits the NF-κB signaling pathway by inhibiting the phosphorylation of Iκ-Bα upstream of the NF-κB signaling pathway. After being pre-treated with TNF-α (10 ng/mL) for 30 min, HeLa cells were treated with [Cu(PMPP-SAL)(EtOH)] (7.5 μg/mL) for 3 h and 6 h, followed by detection of expressions levels of Iκ -Bα and P-Iκ-Bα via western blot. As shown in Fig. 6D (Fig. 1B), similar to the results from a study conducted by Kou Chun 15 , which found that the compound had a relatively low acute toxicity in mice. We also found that the inhibition of cancerous cell growth was due to increased apoptosis induced by [Cu(PMPP-SAL)(EtOH)] treatment. Apoptosis, also known as active death process, is an active and gene-controlled process of cell death for maintaining the stability of the internal environment. At present, there are two main apoptotic signaling pathways, which are the intrinsic mitochondrial pathway and extrinsic death receptor pathway 21,22 . In the mitochondrial www.nature.com/scientificreports/ pathway of apoptosis, decrease in mitochondrial membrane potential promotes the release of Cytochrome c (Cyt c) from the mitochondria into the cytoplasm and activated caspase-3, caspase-7, and caspase-9. Therefore, caspase-9 is an important marker of mitochondrial apoptotic pathway 22 . A study found that, a new copper (II) complex with coumarin derivatives promoted caspase-3 activity and induced apoptosis in lung cancer cells 23 . Cu (II) schiff base complexes are believed to promote HeLa cell apoptosis by activating the mitochondrial apoptosis pathway via the activation of caspase-3 and caspase-9 24 . It is possible that upon treatment of HeLa cells with the [Cu(PMPP-SAL)(EtOH)] compound, the activity of caspase-3 and caspase-9 in cells increased threefold. After pretreatment of HeLa cells with caspase pan-inhibitor (Z-VAD-FMK), followed by [Cu(PMPP-SAL)(EtOH)] treatment, significant reduction in the growth inhibition of HeLa cells was observed. After treatment of HeLa cells with [Cu(PMPP-SAL)(EtOH)], the reduced potential of inner mitochondrial membrane may also have caused the breakdown of PARP protein into activated smaller fragments, and may have resulted in lowering the expression of Bcl-2 protein, a Bcl-2 family protein pathway-associated protein, as well as increased expression of Bax protein. The above results suggest that, [Cu(PMPP-SAL)(EtOH)] induces apoptosis in HeLa cells through the caspase-dependent mitochondria-mediated pathway, thereby leading to inhibition of cell growth. Apoptosis is modulated by complex pathways that involve a series of biochemical regulators and molecular interactions. Early experiments suggested the possibility of interaction between the MAPK pathways and the NF-κB transcriptional cascade 25 . Cell nuclear factor NF-κB is associated with immune responses to cell proliferation, apoptosis, infection, and inflammation 26 . An earlier study had reported that NF-κB in mitochondria might play a regulatory role in cell growth and apoptosis 27 . The activation of NF-κB in tumor cells has been reported to be involved in cell proliferation, blocking of tumor cell apoptosis, promotion of angiogenesis, and enhanced invasion and metastatic capacity of the tumor 28 . In the present study, we found that, [Cu(PMPP-SAL)(EtOH)] exhibited a growth-inhibiting effect on the proliferation of TNF-α-stimulated cervical cancer cells (Fig. 6A). Moreover, we found that [Cu(PMPP-SAL)(EtOH)] inhibited the NF-κB signaling pathway by blocking the Iκ-Bα phosphorylation upstream of the NF-κB signaling pathway (Fig. 6C,D), suggesting that [Cu(PMPP-SAL)(EtOH)] induced cell apoptosis may be related to NF-κB signaling pathway.

Scientific Reports
MAPK signaling pathway plays an important role in regulating biological mechanisms such as cell proliferation and apoptosis 29 . In the PI3K/AKT signaling pathway, the balance between cell proliferation and apoptosis could be modulated by regulating AKT expression, so as to inhibit tumor cell growth 30 . P38/MAPK signaling pathway has been associated with the induction of apoptosis via various stress signals such as TNF-α, interleukin-1, ultraviolet radiation, hyperosmotic stress, and chemotherapeutics 31 , while the activation of JNK pathway might enhance caspase-3 activity, playing an important role in apoptosis 32 . In the present study, we found that, after treatment of HeLa cells with [Cu(PMPP-SAL)(EtOH)], the PI3k/AKT survival signaling pathway was blocked and the P38/MAPK and JNK/MAPK signaling pathways were activated (Fig. 5E,F), suggesting that [Cu(PMPP-SAL)(EtOH)] may induce cell apoptosis by inhibiting PI3K/AKT and activating P38/MAPK and JNK/MAPK pathways.
In summary, the present study demonstrated that, [Cu(PMPP-SAL)(EtOH)] inhibited the growth of human cervical cancer HeLa cells in vitro and induced apoptosis of HeLa cells through the caspase-dependent mitochondria-mediated pathway. The mechanism of apoptosis may be related to the inhibition of NF-κB and PI3k/ AKT pathway, and activation of P38/MAPK and JNK/MAPK signal transduction pathways. These findings showed that, [Cu(PMPP-SAL)(EtOH)] may serve as a potential drug candidate for cervical cancer treatment.
Clonogenic assay. HeLa cells were seeded in 6-well plates at a density of 1 × 10 3 cells/well in the growth medium and cultured for 24 h. Cells were incubated for 24 h with different concentrations (0, 2.5, 5, 7.5 μg/mL) of [Cu(PMPP-SAL)(EtOH)]. Then cells were washed three times with PBS and incubated with DMEM (high glucose) medium for 4 days. After being formed, colonies were washed with PBS and colonies of fewer than 20 cells were discarded. Then each well was added 1 mL of methanol to fix the colonies for 20 min, and 0.1% crystal violet (95% absolute ethanol + 5% PBS) for 15-20 min to stain. Finally, colonies were washed with PBS three times, and the results were scanned with the scanner. To further determine whether the inhibitory effect [Cu(PMPP-SAL)(EtOH)] on HeLa cells growth was affected by caspase pathway, HeLa cells were seeded at a concentration of 1 × 10 5 cells/mL in a 96-well plate, and 5 μM caspase pan-inhibitor (Z-VAD-FMK) was added to each well. After incubation for 0.5 h, HeLa cells were treated with [Cu(PMPP-SAL)(EtOH)] and continued to be cultivated for 24 h. Then cells were incubated with 20 μL of MTT for 4 h, and the OD at 570/655 nm were detected by microplate reader.
Determination of mitochondrial transmembrane potential (∆Ψm). HeLa cells were seeded in 6-well plates and incubated with different concentrations of [Cu(PMPP-SAL)(EtOH)] (2.5, 5, 7.5 μg/mL) for 24 h or 36 h. Then cells were washed with PBS, and incubated with 100 μL of rhodamine 123 (final concentration is 1 μM) for 15 min in the dark. Excess dye was removed by washing with PBS, and measurement of cellassociated fluorescence was performed on FACSCalibur flow cytometer using 488 nm excitation with 505 nm bandpass emission filters.
Western blot analysis. Apoptotic markers such as Bcl-xl, Bax, Bcl-2, PI3K, P38 and JNK were examined by western blot. Shortly, 1 × 10 6 HeLa cells were uncovered with [Cu(PMPP-SAL)(EtOH)] or DMSO for 24-h incubation. Then, the proteins were extorted from cells using RIPA buffer. The protein concentration was measured using Pierce™ Detergent Compatible Bradford Assay Kit (USA). Then, the proteins were positioned to 15% SDSP-PAGE and the gel was reassigned to PVDF membranes. The membrane was blocked with 5% bovine serum albumin for 3 h. The membranes were combined with a suitable primary antibody and kept for 24-h incubation at 4 °C. PARP, Cyt c, Bcl-2, Bax and β-actin antibodies were from Cell Signaling Technology Co. JNK, phospho-JNK, IκBα, p38, phospho-p38, phospho-AKT and AKT antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Then membranes incubated with suitable conjugated horseradish peroxidase secondary antibodies for 1 h. Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology Co. Protein bands were detected using the ECL assay kit (Beyotime, Jiangsu, China) and exposed using a Kodak medical X-ray processor (Kodak, USA).
Statistical analysis. Data were presented as mean ± standard deviation (SD) of three individual experiments. Student t-tests were performed using the SPSS 17.0, and P < 0.05 was considered statistically significant.

Data availability
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.