The Effect Of P8 In DHA-induced Apoptosis In Cancer Cells

Objectives:Dihydroartemisinin (DHA), a main active metabolite of artemisinin, which is making a crucial contribution to the therapy of malaria. In recent years, DHA has been demonstrated to exert preferentially cytotoxic effects toward various human cancers. However, it has been reported that DHA alone is not effective enough for cancer therapy. It has been reported that the stress-inducible protein p8 is involved in the cancer progression and cancer chemoresistance, and we have found the DHA-induced upregulation of p8 attenates the sensitivity to DHA in tumor cells. Thus we focused on the mechacism of p8-induced resistance to DHA in cancer cells, aimed to provide new strategies for cancer therapy.Methods:Two cancer cell lines, human cervical carcinoma cell line HeLa and hunman colon carcinoma cell line HCT116, were used to research the mechacism of p8-induced resistance to DHA in cancer cells. (1) Western Blot were used to detect the protein levels of p8、ATF4、CHOP、caspase3、PARP and LC3. (2) RT-PCR were used to detect the mRNA levels of p8、ATF4 and CHOP. (3) SRB assay was used to detect the cytotoxicity of drugs. (4) siRNAtransfection was used to silence the expression of p8、 ATF4, CHOP and Atg5. (5) pccL-p8 plasmid transfection was used to overexpress p8. (6) Acridine orange (AO) and Dansylcadaverine (MDC) were used to evaluate the abundance of autophagic vacuoles in the cells. (7) Cells undergoing apoptosis were dectected using the Annexin V-FITC/PI apoptosis detection kit (Invitrogen) or Annexin V-PE/7-AAD apoptosis detection kit. (8) Human cervical cancer HeLa xenografts were established to evaluate the in vivo activity of drugs.Results:(1) Dihydroartemisinin upregulates p8 at transcription level and protein level in cancer cells.After the treatment of 20μM DHA, the time-dependent increases in p8, ATF4 and CHOP were observed in both two cell lines. Likewise, by using real-time quantitative PCR, we confirmed that DHA upregulates the mRNA levels of p8, ATF4 and CHOP. To compare with the negative-knockdown cells, the levels of ATF4 and CHOP significantly decreased after DHA exposure in p8-knockdown cells and the identical results were obtained in mRNA levels. Conversely, when cells were transfected with p8-overexpress plasmid, the ATF4 and CHOP express levels further increased than the negative control cells after DHA treatment. Taken together, these data demonstrated that DHA-induced increases of ATF4 and CHOP were mediated by p8 upregulation.(2) p8 upregulation decreases the sensitivity of cancer cells to dihydroartemisinin.HeLa and HCT116 cells were treated with a series of concentration of DHA for 48h, and after that we figured out the survival functions by SRB assays. The survival functions decreased when p8 was silenced by siRNA, whereas the survival functions increased in p8 overexpressed cells compared with the negative control cells. The same results were observed when we detected apoptosis levels by Western Blot and flow cytometry ananlysis. These results confirmed that the higher expression of p8, the lower effect of DHA performs in tumor inhibition.(3) p8 upregulation is involved in dihydroartemisinin-induced autophagy in cancer cells.In negative-knockdown HeLa cells, treatment with 20μM DHA resulted in the formation of red fluorescent AVOs, whereas the DHA-induced AVOs were reduced in p8-knockdown HeLa cells. The same results were obtained in DMC staining, indicating p8 knockdown reverses DHA induced autophagy. Moreover, the LC3-Ⅱ accumulations induced by DHA were reduced when p8 was silenced by siRNA. Conversely, the LC3-Ⅱ accumulations were further increased after DHA treatment when p8 was overexpressed. We discovered similar results when the ATF4 or CHOP was silenced by siRNAs, indicating the p8-ATF4-CHOP axis plays a critical role in DHA induced autophagy in tumor cells.(4) Inhibition of autophagy increases apoptosis in dihydroartemisinin-treated cancer cells.We pretreated with 3-MA or CQ, or silence Atg5 to inhibit autophagy, and then detected the apoptosis rate in HeLa cells that were treated with DHA. And we found that the DHA-induced apoptosis levels were increased after autophagy inhibition, indicating the upregulation of p8-ATF4-CHOP axis induces autophagy to attenuate apoptosis in DHA treatment.(5) The combination of dihydroartemisinin and chloroquine exerted synergistic anti-tumor efficacy both in vitro and in vivo.The combination of DHA and CQ notably decreased the survival function in both HeLa and HCT116 cells, and the CI values indicated that DHA and CQ exerted synergistic anti-tumor efficacy in vitro. In Hela xenografts model, the combination therapy exhibited distinct tumor growth inhibition, with a significant greater extent than DHA-, CQ-or vehicle-treated mice.Conclusions:In summary, for the first time, we found the stress-inducible protein p8 is upregulated rapidly by DHA treating in tumor cells. Furthermore, we demonstrated that the upregulation of p8 induces ATF4 and CHOP expression, and consequently leads to autophagy, which was confirmed to attenuate apoptosis in DHA treatment. In addition, the combination therapy of DHA and CQ showed well synergistic antitumor effect in both vitro and in vivo. These results provide evidence to support to use p8 as a cancer therapeutic target, and suggest that the combination treatment of DHA and CQ might be an effective cancer therapeutic strategy.