Studies On The Cytotoxicity And Antioxidant Activities Of A New Derivative Of Jasmonates

Methyl jasmonate (MJ) is an important member of the plant stress hormones family of jasmonates and shows significant anticancer activity.In the present study, methyl 5-chloro-4,5-didehydrojasmonate (J7),a new derivative of methyl jasmonate, was investigated for its anticancer activity in vitro.Cytotoxicity assay showed that J7 significantly inhibited the growth of HeLa cells and its IC50 value was about 15μM; morphological observation displayed typical apoptotic patterns such as cell shrinkage and cell rounding up;Annexin V-FITC/PI dual staining assay and DNA content analysis results supported further that J7 was able to induce apoptosis of HeLa cell and influence cell cycle process resulting in S and G2 phases arrest.To gain clear insight into the mechanisms of J7-induced apoptosis, the expression levels and activities of the proteins involved in apoptosis-----Bcl-2,caspase-9 and caspase-3 were detected by western blot assay. The result demonstrated that J7-induced apoptosis of HeLa cells was via caspase-dependent mitochondrial pathway in dose-dependent matter. In order to clarify whether MAPK family was involved in regulating J7-induced apoptosis in HeLa cells, the activation of SAPK/JNK and p38 MAPK were also detected. Western bolt results indicated that J7 induced the phosphorylation of SAPK/JNK and p38 MAPK. Therefore, the sub-MAPK pathways of SAPK/JNK and p38 were involved in J7-induced HeLa cell apoptosis.Apoptosis can be triggered by multiple mechanisms, including DNA damage.In the present study, we hypothesized that J7-induced apoptosis of HeLa cells might correlate with its induction of DNA damage.Results from single-cell gel electrophoresis (comet assay) showed that J7 induced DNA single strand breaks before apoptosis induction. Therefore, J7 might induce apoptosis of HeLa cell by induction of DNA damage.J7 arrested cell cycle in S phase.This blocking effect may be associated with the inhibition of DNA replication. In the present study, we investigated the action of J7 on DNA replication. [3H]deoxythymidine triphosphate([3H]dTTP) incorporation assay demonstrated that J7 decreased the incorporation of [3H]dTTP in cellular DNA replication although cell number was not significantly decreased after J7 treated. Therefore, J7 was able to inhibit DNA replication. To known the mechanism of the action of J7 on DNA replication, the activities of topoisomerase I (topo I) and replication protein A (RPA) were detected. The result showed that 1.0 mM of J7 started to show inhibition of the DNA relaxation activity of topo I.However,15μM of J7 could not inhibit topo I activity. In the range of 10-40μM, J7 had no influence on RPA's ssDNA binding activity. Therefore, topo I and RPA were not the target molecules involved in J7-induced S phase arrest.Among the possible causes of cancer, damage to DNA and other molecular molecules by ROS,ranks high as a major culprit in the onset and development of the disease. Therefore, in the development of anticancer drugs, the antioxidant activity of the target compounds is evaluated to get further insight into the mechanism of their actions.In our study, DPPH free radical scavenging assay, superoxide radical scavenging assay (NBT) and measurement of Trolox Equivalent Antioxidant Capacity (TEAC) assay were performed to evaluate the antioxidant activity of J7.DPPH free radical scavenging and NBT assay showed that J7 had weak free radical scavenging activity which was not comparable to that of ascorbic acid; in TEAC assay, J7 exhibited similar ability with ascorbic acid to scavenge ABTS·+free radical. Furthermore, J7 could significantly protect pBR322 DNA from hydroxyl radicals-induced breaks at low concentration (5-10μM).Therefore, we suggested that J7 is a potent anticancer agent, which also can help to prevent occurrence of cancer by alleviating the oxidative stress in cells.