Effect Of Cu-PMPP-SAL On Apoptosis Of HeLa Cells And The TNFα-induced NF-κB Signaling Pathway

Metal complexes have been considered as potential sources of anticancer drugs for years. Some platinum-based anticancer drugs such as cisplatin, carboplatin, and oxaliplatin have been widely used in clinical. However, these drugs always accompany with serious side effects and drug resistance. Therefore, it is urgent to develop anticancer drugs with high effectivity and low toxicity. The 4-acyl pyrazolone derivatives, especially the Schiff based derivatives, are useful organic ligands for construction of novel complexes with diverse structures due to the multiple active coordination sites and the tautomeric effect of enol form and keto form. Great attention has been paid to pyrazolone-based metal complexes due to the advantages including stability, easy synthesis, easy alteration, and abundant bio-activity of antibacterial, antiviral and anticancer.Nuclear factor-kappa B(NF-κB) is a transcription factor. And dysregulation of the pathway is associated with diseases such as inflammation and cancer. Since the NF-kB pathway has been reported to be constitutively active in cancer cells such as breast, colon, and melanoma, the inhibition of this pathway may be useful in the treatment of tumors. Tumor necrosis factor alpha(TNFα) is inducing factor of apoptosis in tumor cells. However, several recent studies have reported that TNFα may activate the transcription activity of NF-κB, and in turn, this causes the tolerance of cancer cells to the chemotherapeutic drug-induced cytotoxic effects. But up to now, the effective of pyrazolone compounds on the targeting blockage of NF-κB pathway in HeLa cells have not been reported.In this paper, the anti-tumor effective of 25 kinds of pyrazolone derivatives were screened. It is investigated that the effect of Cu-PMPP-SAL to inhibit the proliferation of cancer cells in vitro and in vivo, induce cell apoptosis, and block the TNFα-induced NF-κB signaling pathway. Also, the preliminary antitumor mechanism and drug toxicity of Cu-PMPP-SAL were measured. The results would provide basic information for the structure optimization of new pyrazolone metal complex and the application and development of anticancer drugs. The main results of this study were as follows:(1) Cu-PMPP-SAL, Cu-PMPP-TAH, Cu-PMBP-TAH, Cu-PMPP-nSAL had good anti-tumor activity, IC50 values were 2.082 μg/mL, 1.287 μg/mL, 1.186 μg/mL, 2.559 μg/mL, respectively, when treated HeLa cells 72 h with pyrazolone derivatives by MTT assay. In addition, we also found that Cu-PMPP-SAL had a good anti-tumor effect of HeLa and Eca-109 cells, which was worth the further study to its mechanism of action.(2) Cu-PMPP-SAL had a good inhibition effect of HeLa, and it promoted the induction apoptosis in HeLa cells through caspase-dependent mitochondria Pathway. Cu-PMPP-SAL-treated HeLa cells showed typical apoptotic features, including condensed fragmented nucleus and gather in the edge of nuclear membrane by Hoechst 33258 staining and TEM. And LDH activity assays indicated that the LDH release rate of the cells increased, suggesting the cell membrane damaged by compound. PI staining assays showed the cell cycle arrest in S phase. Cu-PMPP-SAL induced apoptosis was assessed by Annexin V-FITC/PI staining(P<0.05). Cu-PMPP-SAL promoted the loss of ΔΨm and caspase-3 and-9 activity(P<0.05). Furthermore, Cu-PMPP-SAL induced cytochrome c release from mitochondria, PARP cleavage, up-regulation of Bax and down-regulation of Bcl-2 in HeLa cells was detected by Western blot. In summary, Cu-PMPP-SAL promoted the induction apoptosis in HeLa human cervical carcinoma cells through caspase-dependent mitochondria Pathway.(3) Cu-PMPP-SAL inhibited NF-κB signaling pathway through inhibited the phosphorylation of Iκ-Bα. To observe cell viability, MTT assay werecarried out in HeLa cells with Cu-PMPP-SAL treatment after TNF-α stimulated. The effect of Cu-PMPP-SAL to TNF-α induced NF-κB signaling pathway was assessed by NF-κB luciferase reporter. Expression of the proteins related with NF-κB was detected by Western blot. The results showed that Cu-PMPP-SAL could inhibited the proliferation of HeLa cells which activated by TNFα. The result of double luciferase reporter gene was shown that Cu-PMPP-SAL inhibited TNFα-induced NF-κB activation, and the result of Wastern blot confirmed that Cu-PMPP-SAL might be inhibited the phosphorylation of IκBα, the protein of NF-κB signaling pathway upstream, then inhibited NF-κB signaling pathway. These data suggest that Cu-PMPP-SAL inhibited NF-κB signaling pathway through inhibited the phosphorylation of Iκ-Bα.(4) Cu-PMPP-SAL promoted the induction apoptosis in B16 cells in vitro and in vivo. And Cu-PMPP-SAL can inhibit the growth of B16 solid tumor by inhibited angiogenesis in vivo. Cu-PMPP-SAL significantly induced cell viability changes in a dose-dependent manner in B16 cells was assessed by MTT(IC50: 4.021 μg/mL), Cu-PMPP-SAL-treated B16 cells showed typical apoptotic features, including condensed fragmented nucleus by Hoechst 33258 staining. PI staining and Annexin V-FITC/PI assays showed the cell cycle arrest in G0/G1 phase and the apoptosis rate significantly increased when compared with the control group(P<0.05), respectively. Growth rate of tumor in mice model was also significantly inhibited in Cu-PMPP-SAL therapy group as compared with the control grouop(P<0.05). The inhibition rates of concentration dose group(30 mg/kg, 20 mg/kg, 10 mg/kg) were 54.8 %, 44.44 % and 35.58 %, respectively. The tumor tissue cells had different levels of necrosis in high and medium concentration dose group. The liver, lungs and other organs in C57 BL/6J mice haven’t significant pathological damage. The positive expression indexes of highly glycosylated i transmembrane glycoprotein(CD34), vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF) in high and medium concentration dose group were significantly lower than ones of CD 34, VEGF and bFGF in control group(P<0.01). In summary, Cu-PMPP-SAL promoted the induction apoptosis in B16 cells at time and dose-dependent mannerin in vitro. And Cu-PMPP-SAL can inhibit the growth of B16 solid tumor by inhibited angiogenesis in vivo.(5) Cu-PMPP-SAL had the smaller acute toxicity on mice, suggesting that it has better security. The results of acute toxicity tests showed that the LD50 value of intraperitoneal injection of Cu-PMPP-SAL was 33 mg/kg, 95% confidence range was 25.5- 43 mg/kg. The mice in high dose-group(200 mg/kg, 400 mg/kg and 800 mg/kg) showed breathing heartbeat, seizures and other symptoms. Mortality of mice in every group presented in positive relationship with the dosages of Cu-PMPP-SAL, Cu-PMPP-SAL had better security which compared with positive drug dose groups(cisplatin, DDP, 12.5 mg/kg).In summary, Cu-PMPP-SAL promoted the induction apoptosis in HeLa human cervical carcinoma HeLa cells through caspase-dependent mitochondria Pathway. Cu-PMPP-SAL inhibited NF-κB signaling pathway through blocked the phosphorylation of Iκ-Bα. In addition, Cu-PMPP-SAL promoted the induction apoptosis in B16 cells at time and dose-dependent mannerin in vitro. And Cu-PMPP-SAL can inhibit the growth of B16 solid tumor in vivo. In addition, the compound had the smaller acute toxicity on mice, suggesting that it has better security.