Ph Ii-7 Mode Of Action And Mechanism

Although concepts and technologies have evolved greatly on cancer therapy,but most clinical cancer treatments still rely on chemotherapy,especially for metastasis cancer and haematological malignancies.The major obstacle to chemotherapy is that tumor cells treated with chemotherapy agents commonly develop resistance to a variety of structurally and mechanistically unrelated drugs,the phenomenon is called multidrug resistance(MDR).In previous study,from traditional Chinese medicine Danggui Longhui Wan,to indigo naturalis,from indigo naturalis to indirubin,in previous study, the activity of Danggui Longhui Wan was traced more and more precisely.Our lab identified the active subunit of,and synthesized a series of derivatives based on the active subunit and screened out the PHⅡ-7.The MDR cell lines was highly resistant to ADM,the resistance fold of K562/A02, MCF7/ADR and HL60/ADR compared with their parental cell lines are 93.93,25.16,182.3 folds respectively,which explains the trouble caused by MDR phenomenon in clinical chemotherapy.As to PHⅡ-7 the IC50 values had no significant difference or much lower difference than ADM between the cell lines and their corresponding MDR sublines, suggesting that PHⅡ-7 is highly effective against MDR phenotype cells compared with ADM.we applied genomic and chemical approaches in researches related with ancient traditional Chinese medicine.To unravel the action mode of PHⅡ-7,we analyzed the genome profiling changes in PHⅡ-7 treated K562 and K562/A02 cells.The result guided our study to two aspects affected by PHⅡ-7:its regulation of MDR1 gene expression, and its influence on apoptosis and cell cycle.A dramatic decrease in ADM attainment in K562/A02 cells than in K562 cells was illustrated by CSLM imaging,comparatively,the intracellular intention of FITC labeled PHⅡ-7 did not significantly vary between the K562 and K562/A02 cells,the PHⅡ-7 molecules just redistributed into nucleus time dependently,rather than being pumped out of the cells like ADM,providing direct evidence that PHⅡ-7 is not influenced by drug efflux mediated MDR mechanism.Very low concentration(＜IC10)PHⅡ-7 pre-treated K562/A02 cells showed attenuated drug efflux,time and dose dependently.Genome profiling indicated that the MDR1 expression level significantly decreased after the treatment,which confirmed that PHⅡ-7 inhibited MDR1 expression.Meanwhile,the expression level of protein kinase C alpha(PKCA) substantially decreased.We further analyzed the relationship between the expression of the two genes by knockout of PKCA expression by RNAi,which resulted in significant reduction in MDR1 expression, indicating that PHⅡ-7 impeded MDR1 expression by inhibition of PKCA transcription.We also found substantially decreased expression of c-FOS and c-JUN,in PHⅡ-7 treated group and RNAi group.PHⅡ-7 treated K562/A02 cells significantly decreased the expression of PKCA,c-FOS,c-JUN,and MDR1,which is detected by Genome profiling, RNAi knockout of PKCA expression in K562/A02 cells also resulted in salient reduction in expression of c-FOS,c-JUN and MDR1,which is consistent with the result of PHⅡ-7 treatment,indicating a positive correlation between PKCA,AP1 and MDR1 expression.According to our data,100nM ADM induced significant apoptosis in K562 cells,but failed to do so in K562/A02 cells.Notably K562 and K562/A02 cells treated with PHⅡ-7 were induced apoptosis without considerable difference at three concentrations, indicate that PHⅡ-7 is capable of overcome apoptosis resistance and induce significant apoptosis in K562/A02 cells.Genome profiling and gene expression analysis shed light on the possible underlying mechanism of PHⅡ-7 induced apoptosis,The apoptosis related gene PML(promyelocytic leukemia) were significantly up-regulated,PML is essential for multiple apoptotic pathways.Analysis of mice and cells lacking PML has demonstrated that PML is involved in various apoptotic pathways.Previous studies have demonstrated that certain indirubin derivatives are capable of inducing cell cycle arrest in cancer cells.Our data indicate that PHⅡ-7 substantially inhibit cell cycle of both K562 and K562/A02 cells in S phase.Cells inhibited in S phase are not able to enter G2 and M phase,thus were prohibited from mitosis and further proliferation.The temporal order of many critical cell cycle events must be strictly maintained to ensure cell survival and integrity.A simple example is that of genome duplication which must be completed before cell division.Our genome profiling data revealed several PHⅡ-7 responsive genes are related with cell cycle control,such as HBP1,CHK1 and P21.The findings were confirmed by RT-qPCR gene expression analysis,expression level of CHK1 increased after PHⅡ-7 treatment in both K562 and K562/A02 cells.CHK1 is apparently necessary for an intra-S phase checkpoint,ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.Increased P21 expression also contributes to S phase arrest.High p21 producing cells move much more slowly through S phase,p21 protein can elevate E2F-1 activity to arrest a substantial subset of cells in S phase.Judging by our data,PHⅡ-7 substantially induced apoptosis and S phase cell cycle arrest in MDR tumor cells,by regulation of specific gene expression,and finally resulted in effective inhibition and termination of MDR tumor growth,it is a promising lead compound for anti-MDR tumor drug development,and may represents a new solution for MDR phenomenon which is still the major cause for tumor therapy failure and patient death.Our way of discovering PHⅡ-7,represents an effective mode of lead compound discovery based on traditional Chinese medicine. Objective:to investigate the function of sorcin in multidrug resistance in MDR human leukemia cell line K562/A02 and to uncover the mechanisms and signal transduction pathways related.Methods:sorcin-targeting small interfering RNA was successfully designed and synthesized to inhibit the expression of sorcin in K562/A02 cells.The inhibition of sorcin was validated by realtime RT PCR.Then,the global gene expression profile was tested by Affymetrix U133 plus 2.0 microarray and the differences in gene expression was obtained comparing to K562/A02 cells untreated.Results:knock-down of sorcin induced variation in the expression level of a series of genes.Including c-FOS,PDGF-C and IL-8.Conclusion:Sorcin may influence the multidrug resistance phenotype by altering the transcription level of c-FOS,PDGF-C and IL-8.