The Ethyl Acetate Component Of The Big Blond Cockroach Induces Apoptosis Of Leukemia Cells And Synergistically Enhances The Anti-tumor Mechanism Of Doxorubicin

Background and ObjectivePolytrichum commune L.ex Hedw ?PCLH?, belonging to the genus Polytrichaceae, is used as a Traditional Chinese Medicine to stop bleeding and treat pneumonia, fever, hemostatic, traumatic injury and uterine prolapse, etc. Recently, PCLH has been confirmed with the broad-spectrum antineoplastic activity by modern pharmacology study. The proliferation of cancer cells including carcinoma of breast, esophagus, colon, and leukemia etc. can be inhibited effectively by PCLH, among which leukemia is the most affected one. The ethyl acetate fraction ?PC-EEF? is the main active component of PCLH, while the molecular mechanism of which that inhibits the proliferation of leukemia has not yet illuminated. Besides, the function of PC-EEF on human multidrug-resistant leukemic cell line has not yet been investigated.In the present study, we investigated the effect of PC-EEF on human leukemic cell lines K562 and K562/DOX. The aim of this study was summarized as follows:1. PC-EEF induces apoptosis of K562 and K562/DOX cells and its mechanisms2. PC-EEF synergistically enhances the anti-tumor effect of DOX on K562/DOX cells:a role for oxidative stress-mediated cell damage and PI3K/Akt pathway inhibition.Methods and Results1. The phytochemical analysis of PC-EEF The spectrophotometry and high performance liquid chromatograph ?HPLC? were used to analyze the chemical compositions of PC-EEF qualitatively and quantitatively. ? Flavonoids were the main bioactive constituents of PC-EEF and the content was 88.85 ± 0.89%. ? Typical chromatogram of HPLC indicated that PC-EEF displayed 9 main peaks and included two peaks with the same retention time of rutin ?9.2 min? and luteolin ?13.3 min?, respectively. The concentrations were as follows:rutin ?4.77 ± 0.04%? and luteolin ?6.82 ± 0.06%?.2. PC-EEF induces apoptosis of K562 and K562/DOX cells and its mechanisms In the study, it is very interesting for us to evaluate the apoptotic responses to PC-EEF treatment in human leukemia K562 and K562/DOX cells. Following PC-EEF treatment, hallmarks of apoptosis were detected by morphological observation, biochemical analysis and molecular measurements. The relationship between Ca²⁺ and apoptosis was further obtained by applying pharmacological inhibition studies. The potential mechanisms of PC-EEF induced cellular responses were also evaluated. The main research methods and results are as follows:? MTT assay revealed that PC-EEF could selectively inhibit K562 and K562/DOX cells viability in a dose- and time-dependent manner with the IC50_24h of 10.71 ± 0.09 ?g/ml and 16.98 ± 0.14 ?g/ml, respectively. However, rutin and luteolin showed low toxicity on K562 cells. Under the same conditions, PC-EEF nearly had no impact on the growth of human peripheral blood mononuclear cells ?PBMCs?. Besides, colony-formation assay and EdU incorporation assay further confirmed the impact of PC-EEF on the K562 cell proliferation.? Based on previous study, this study was to analyze the effect of PC-EEF on the cell membrane in K562 and K562/DOX cells. The ultrastructural changes of the PC-EEF-treated cancer cells were identified by scanning electron microscope observation. Membrane integrity loss and membrane depolarization occurred in the early step of cell damage following PC-EEF treatment, and gradually increased with the incubation dose and time, indicating the alteration of plasma membrane structure and function might be related with the growth inhibition and cell death.? After PC-EEF treatment, an elevation of intracellular Ca²⁺ level occurred as early as 4 h and further increases continued for at least 24 h. Wherein, intracellular Ca²⁺ concentration reached the highest level at 8 h. In order to investigate the role of Ca²⁺ in PC-EEF-induced cell death, K562 cells were pre-incubated with BAPTA-AM followed by PC-EEF treatment.1 ?M BAPTA-AM nearly completely reverses the PC-EEF-induced intracellular Ca²⁺ increases. Meanwhile, BAPTA-AM pretreatment almost completely abolished the cytotoxicity and reversed the cell membrane integrity loss, suggesting the potential role of Ca²⁺ in PC-EEF-induced cell membrane damage as well as the resultant cell death.? As the immunofluorescence, FACS analysis and western blotting analysis revealed, after PC-EEF treatment, apoptotic features such as mitochondrial membrane potential collapse, cytochrome c release and Bcl-2/Bax down-regulation were prominent and time dependent. Consistent with the findings, PC-EEF treatment resulted in significant activation of Caspase 3, PARP cleavage and apoptosis, which suggested mitochondria dependent apoptosis pathway was involved.?5? The presence of BAPTA-AM almost completely restore the dissipation of mitochondrial membrane potential and cell apoptosis. The findings implied perturbations in intracellular Ca²⁺ homeostasis might be the upstream event associated with mitochondria dysfunction and subsequent apoptosis events.3. PC-EEF synergistically enhances the anti-tumor effect of DOX on K562/DOX cells:a role for oxidative stress-mediated cell damage and PI3K/Akt pathway inhibition In the current study, we will evaluate the feasibility and the advantage of the combination of PC-EEF with DOX in K562/DOX cells, and its action mechanism. The main research methods and results are as follows:? MTT assay revealed that PC-EEF elicited a significant cytotoxicity against K562/DOX cells with resistance factor ?RF48h=1.91?, which was much lower than that of DOX ?RF48 h=131?. PC-EEF and DOX have a significant synergism and the combination index ?CI? was less than 1. PC-EEF dose dependently increased the sensitivity of K562/DOX cells to DOX. The combination of 9 ?g/ml PC-EEF with DOX showed more potent killing effects on K562/DOX cells than 1 ?M LY 335979 with reversal index ?RI? 14.98 versus 6.62. These data indicated that 9 ?g/ml PC-EEF could be applied combining with DOX to get better anti-tumor activity. Guava Viacount assay further confirmed the significant synergism.? Apoptosis was analyzed using Annexin V-FITC/7-AAD staining. A positive enhancement ?27.85 ± 0.22%,57.60 ± 0.47%? on apoptotic cell death was detected at 48 h after the combined treatment with PC-EEF and DOX.? Fluorescence microscopy revealed that PC-EEF could obviously suppress Rhodamine-efflux and increase DOX-uptake, but did not affect the cell membrane integrity at the early stage. The potency of PC-EEF was comparable with that of LY 335979. Meanwhile, the immunofluorescence demonstrated the significant decreased expression level of P-gp after the combined treatment with PC-EEF and DOX. Besides, western blotting analysis revealed that PC-EEF and DOX synergistically inhibited the expression level of p-PI3K and p-Akt. Therefore, we speculated that the inhibition of PI3K/Akt signaling might play a pivotal role in sensitizing the anti-tumor effects of DOX, presumably through the inhibitory effects on its downstream target P-gp expression indirectly.? As the immunofluorescence and FACS analysis revealed, a combination of PC-EEF with DOX exhibited more effects in promoting the level of ROS generation, the increase of y-H2AX foci formation and the dissipation of mitochondria potential. Consistent with the findings, co-treatment resulted in significant increase in phosphorylation of H2A.X and PARP cleavage. In addition, PC-EEF and DOX synergistically decreased the ratio of Bcl-2/Bax.? The inhibitor studies suggested that the presence of ROS scavenger NAC significantly decreased ROS generation. Meanwhile, NAC also visibly reduced the cell viability loss, DNA damage, mitochondria injury and apoptotic cell death, suggesting the potential role of ROS in combination therapy-induced apoptotic cell death. In addition, pretreatment with NAC significantly attenuated the inhibition of PI3K/Akt signaling and clearly reversed the down-regulation of P-gp expression. The findings further implied the potential role of ROS following the combination of PC-EEF with DOX.ConclusionPC-EEF disrupted the membrane system and mediated apoptosis via a Ca²⁺-dependent mitochondrial pathway. PC-EEF synergistically enhanced the anti-tumor effect of DOX on K562/DOX cells via oxidative stress-mediated cell damage and inactivation of PI3K/Akt pathway.