The Role And Machanism Of Pyrroloquinoline Quinone In Inhibiting Chondrosarcoma Growth

Chondrosarcoma is the second most common primary malignant bone tumor,which is not sensitive to radiotherapy and chemotherapy.Pyrroloquinoline quinone(PQQ)was first recognized as a bacterial enzymatic cofactor,which is a water soluble and heat stable small molecular.PQQ is closely related to health in mammalian system,it can promote the growth and reproduction,also can protect the nervous and cardiovascular system.Recent studies have shown,PQQ has significant anti-tumor effect including cytotoxicity on human lung adenocarcinoma,liver and brain cancer cell lines,and lesser side effects on normal cells,which makes the PQQ may become an ideal drug in cancer therapy.However,whether PQQ has cytotoxicity on chondrosarcoma and its anti-tumor molecular mechanism remains unknown.In order to determine whether PQQ has specific cytotoxicity in bone tumor cell lines,we cultured SW1353(chondrosarcoma cell line),Saos-2(osteosarcoma cell line),WRL68(human hepatocytes),HEK293(human embryonic kidney cells)and XJH(B lymphocytes)cells in vitro,treated with different concentrations of PQQ,and detected the cell death rate.Results showed that mortality rate of tumor cells were increased along with the raise of the concentration of PQQ.There was no significant toxicity to normal cells when PQQ concentration below 120?M.These results demonstrated that PQQ played a role in killing specifically bone tumor cells including SW1353 and Saos-2.In order to identify whether the cytotoxic effect of PQQ on SW1353 cells is time-dependent,we treated SW1353 cells with PQQ in vitro and observed mortality at different time points,the results showed that the death rate of tumor cells was also increased as PQQ treated-time was increased.In order to clarify whether the killing effect of PQQ on tumor cells is through the promoting cell apoptosis,we used flow cytometry to comparative analysis the level of apoptosis in the PBS-treated control and PQQ-treated SW1353 and Saos-2 cells.Results showed that after PQQ treatment,cell apoptosis was increased significantly.These results indicate that PQQ kills the bone tumor cells by inducing tumor cell apoptosis.Previous studies reported that PQQ promoted tumor cell apoptosis by increasing the levels of oxidative stress.In order to determine whether PQQ treatment increase oxidative stress levels in SW1353 cells,and explore relative molecular mechanism,we used flow cytometry,western blot and biochemical analysis to compare the oxidative stress levels and expression of oxidative stress related molecules in PBS-treated control group and PQQ treated groups.Compared with PBS control group,reactive oxygen species(ROS)and hydroxyl radical(OH·)levels were significantly increased in PQQ treated cells.Meanwhile,glutathione levels and total SOD activity were significantly decreased,however,SOD1 and SOD2 protein levels were not altered significantly.These results suggest that PQQ may inhibit the activity of SOD1 and SOD2 protein.As a small molecule,in order to clarify whether PQQ has direct interaction with SOD1 and SOD2 protein to affect their activity,we used molecular docking simulation technology to analyze possible binding protein form of PQQ,SOD1 and SOD2.We found that PQQ can combine the amino acids around the SOD1 or SOD2 active center with three or four hydrogen bonds,to form a stable structure.So PQQ may inhibit the SOD1 and SOD2 protein activity by blocking the active sites.To sum up the results,PQQ induced SW1353 cells apoptosis by increasing the oxidative stress with reduced the activity of SOD1 and SOD2 and GSH levels.To determine whether PQQ induced SW1353 cell apoptosis through the mitochondrial-dependent pathway,we used flow cytometry,western blot and co-immunoprecipitation technique to analyze the changes of mitochondrial membrane potential and the mitochondrial apoptotic pathway related molecules in control cultures and PQQ-treated cultures.Results showed that mitochondrial membrane potential was decreased significantly in PQQ-treated cultures compared with control cultures.Caspase 3 precursor levels were reduced accompany with the increasing concentration of PQQ,which were blocked by Z-VAD pretreatment.The binding of Smac and XIAP was increased significantly,whereas the combination of caspase 3 and XIAP was reduced significantly.Mitochondrial cytochrome C levels were decreased as PQQ concentration was increased,in contrast,cytoplasmic cytochrome C levels were increased significantly.The levels of mitochondrial AIF and EndoG were down regulated when PQQ concentration was increased,in contrast,nucleus levels of AIF and EndoG were increased as PQQ concentration was increased.These results indicate that PQQ induced SW1353 cell apoptosis by activating the mitochondrial-dependent apoptotic pathway.The treatment used combined drugs is one of the basic principles of cancer therapy.It has become a new way for cancer therapy to inhibit TNF induced NF-?B signaling pathway or to promote TNF induced apoptosis.Besides promote apoptosis of tumor cells,it is still unclear whether PQQ has a synergistic effect with TNF?.In order to clarify whether TNFa can promote the PQQ-induced apoptosis in SW1353 cells,we examined the cell death,cell morphology and apoptosis levels in different treated groups,including PBS-,PQQ-,TNF?-,combined PQQ with TNF?-treated groups.Results showed that compared to PQQ-treated group,tumor cells mortality,cytoskeleton damage,nuclei consendation and apoptosis levels were increased significantly in combined PQQ with TNF?-treated group.These results indicate that PQQ and TNF? play a synergistic role in inducing apoptosis of SW1353 cells.In order to determine whether TNF? promoting apoptosis in SW1353 cells induced by PQQ is related to the PQQ inhibition of p65 nucleus translocation caused by TNF?,we used western blot to detect the alterations of localization of p65 protein when SW1353 cells were treated with combined TNF? with different concentrations of PQQ.The results showed that protein levels of p65 in the nuclei were decreased gradually,but increased gradually in the cytoplasm when PQQ concentration was increased.These results suggest that PQQ can inhibit p65 nucleus translocation induced by TNF?.In order to verify whether PQQ inhibition of TNF? induced p65 nucleus translocation was associated with the continuous activation of JNK,the JNK and phospho-JNK levels were examined using in cell Western at different time points in cells treated with TNFa and PQQ.JNK phosphorylation occured 10 minutes after TNFa treatment,and then was suppressed in TNFa treated cultures,however,JNK phosphorylation were kept until 2hours in combined TNFa with PQQ-treated cultures.These results suggest that combined PQQ with TNFa can sustain phosphorylation of JNK.Our results indicate that PQQ inhibited TNFa induced p65 nucleus translocalization to promote apoptosis,at least in part,mediated by sustaining JNK phosphorylation.In order to determine whether PQQ can inhibit tumor growth,we used xenograft model,tumor volume was measured and the cell proliferation and apoptosis were examined in control group and PQQ-treated group by immunohistochemical staining and TUNEL staining,respectively.Results showed that compared with the control group,tumor volume and the percentage of proliferating cell nuclear antigen(PCNA)positive cells were reduced significantly,whereas the percentage of apoptotic cells were increased significantly in PQQ-treated group.These results indicate that PQQ plays a role in inhibiting tumor growth by reducing the proliferation and promoting the apoptosis of tumor cells.To determine whether decreased proliferation and increased apoptosis of transplanted tumor cells induced by PQQ were associated to increased oxidative stress,DNA damage and decreased p65 nucleus translocation,we compared the ROS levels,DNA damage related indicator ?-H2AX and p65 nuclear levels in transplanted tumor cells treated with vehicle or PQQ using flow cytometry and immunohistochemical staining.Compared with the vehicle group,ROS levels,the percentage of y-H2AX positive tumor cells were increased significantly,whereas the levels of nucleus p65 were decreased significantly in PQQ-treated group.These results indicate that PQQ plays a role in inhibiting tumor cell proliferation and promoting apoptosis by inducing oxidative stress,increasing DNA damage and reducing p65 nucleus translocation.Results from this study demonstrated that PQQ plays a role in inhibiting chondrosarcoma growth by reduced the proliferation and induced apoptosis through the mitochondrial-dependent apoptosis pathway by increasing oxidative stress and mitochondrial injury.Furthermore,PQQ also plays a synergistic role in inhibiting chondrosarcoma growth by blocking TNFa-induced p65 nuclear translocation and sustaining JNK phosphorylation.This study provides an experimental and theoretical basis for the application of PQQ or combined PQQ with TNFa together in chondrosarcoma therapy.