Anti-cancer Mechanism Of Novel Reductase Q39Induced The Apoptosis Of K562and Bel-7402in Hypoxia

Objective:Hypoxia is one of the inevitable circumstance of various tumors, which controls a different level of regulation in tumor progression and results in tumor resistance to radiotherapy and chemotherapy. Moreover, Hypoxia-induced factor-la (HIF-la) is a transcriptional complex that is activated in response to hypoxia and growth factors, which plays a central role in tumor progression, invasion and metastasis.3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline-1,4-dioxide (Q39), modified from TPZ, displays a strong activity against human cancer cells both in hypoxia. Thus the advanced research on Q39seems extraordinarily appealing. This study investigate the mechanism of Q39-induced apoptosis of K562and Bel-7402cells in hypoxia.Methods:MTT assay was used to determine the50%inhibitory concentrations (IC50) of Q39on human cancer cells K562and Bel-7402. DAPI staining and flow cytometry was used to determine the apoptosis rate induced by Q39. JC-1staining was used to determine mitochondria membrane potential. Western-blotting was used to determine apoptotic-related protein expression both in normoxia and in hypoxia. Immunostaining was used to detect the HIF-1α translocation after Q39treatment.Results:l.The mechanism of Q39-induced apoptosis of K562cell in hypoxia:1) Q39exhibited markedly antiproliferative activity with IC50values (0.21±0.05) μmol/L in hypoxia.2) DAPI staining showed that cell apoptosis appeared6h after Q39treatment, with cell shrinkage and apoptotic bodies later.3) Flow cytometry analysis showed that K562cells were treated with Q39for0,12,24and48h respectively, the apoptotic rate was4.1%、34.5%、42.5%and60.1%in hypoxia rather than5.5%、5.1%、4.6%and24.4%in normoxia, indicating Q39induced K562apoptosis in a time-dependent manner in hypoxia.4) By fluorescence stain assay, obvious mitochondria membrane potential (△ψm) loss was detected in K562cells in hypoxia after Q39treatment for indicated time.5) Western-blotting assay demonstrated that Q39decreased protein expression of HIF-1α, VEGF, Bcl-2and procaspase-3, meanwhile increased protein expression of Bax, cleaved caspase-3and cleaved PARP, indicating that Q39had great anti-tumor activity in hypoxia and mitochondrial pathway might be involved in the activation of a caspase cascade to induce cell apoptosis. Moreover, we found that Q39downregulated the expression of p-ERK and p-p38and upregulated p-JNK, elucidating that MAPK pathways may also be implicated in suppressing tumor growth.2. The mechanism of Q39-induced apoptosis of Bel-7402cell in hypoxia:1) Q39exerted anti-proliferative effects against human cancer cells Bel-7402in hypoxia, the IC50values was (3.87±0.56) μmol/L in hypoxia.2) Cell apoptosis was induced by Q39treatment in a dose-dependent manner, the apoptotic values were (27.1±4.8)%,(50.2±5.6)%and (62.5±6.2)%induced by Q39(5-20μmol/L) in hypoxia respectively.3) MAPK pathway was activated in hypoxia, the phosphorylated ERK1/2, JNK and p38was increased in hypoxia.4) Q39inhibited the Phosphorylation of ERK1/2rather than p38or JNK in hypoxia, indicateing p-ERKl/2was the major factor involved in Q39-induced apoptosi.5)immunostaing study revealed that HIF-1α translocation was blocked after Q39exposure, menawhile, western blotting showed that HIF-1α expression decreased notably by Q39treatment, indicating that suppression of ERK1/2Phosphorylation by Q39caused reduction of HIF-1α expression, which induced cell apoptosis finally.Conclusions:The present study demonstrated Q39was a novel compound against K562and Bel-7402cells in hypoxia via decrease of HIF-1α expression and induction of apoptosis-associated protein expression. These results will provide new strategy to find novel targets of anticancer drugs in hypoxia and provide new ideas of HIF-1α induced tumor growth.