Mechanism Of Sinoporphyrin Sodium Based Sonodynamic Therapy Induced Apoptosis Of Hepatocellular Carcinoma In Vitro And In Vivo

Hepatocellular carcinoma（HCC）is considered one of the most globally widespread malignancies and the third leading cause of tumor mortality.Surgery is the conventional approach to treat HCC,but only 1/3 of cases are identified at early stages and treatable by surgery due to the high invasiveness and fast development of HCC.The resection rate for HCC is less than 15%,while the postoperative recurrence rate is approximately 50%.These existing problems of current treatment modalities demand the search for new therapeutic methods to achieve more successful intervention.Sonodynamic therapy（SDT）was initiated by Yumita et al in 1989.Similar to photodynamic therapy（PDT）,SDT uses low intensity ultrasound to stimulate sonosensitizers specifically accumulated in carcinoma cells to produce cytotoxic effects.In addition,because the low toxicity of sonosensitizers,SDT can be performed repeatedly without severe side effects,making it especially suitable for elderly and weak cancer patients who cannot undergo surgery or cannot tolerate the body damage of radiotherapy or chemotherapy.The sonosensitizer is the key in the effectiveness of SDT.In practice,many photosensitizers used in photodynamic therapy were used in SDT,but their SDT effect is not ideal because phot-sensitive does not means sono-sensitive.In this study,the mechanism of apoptosis induced by sinoporphyrin Sodium（DVDMS）combined with low-dose ultrasound in human liver cancer cells（Hep-G2）was studied in detail through in vitro and in vivo experiments,and compared with the effectiveness of other traditional sonosensitizers,the potential mechanism of DVDMS as a better sonosensitizer was analyzed.Firstly,the absorption spectrum of DVDMS was measured by multi-volume spectrophotometer.The results show that DVDMS had five absorption peaks,with the maximum peak at 405 nm and the maximum emission peak at 624 nm.The results of cell metabolism showed that the concentration of DVDMS in hepatoma cell Hep-G2 reached the peak after 5 hours of incubation,and remained relatively high after 9 hours,while in normal liver cell HL-7702,the concentration reached the peak after 3 hours,and then decreased rapidly.Therefore,the co-incubation time of DVDMS and cells for 5 hours was selected as the start time of ultrasound treatment.The analysis of cell viability and apoptosis showed that 2.5μg/m L DVDMS combined with 0.5 W/cm² ultrasound（DVDMS-SDT）could achieve the best inhibitory effect.Under these conditions,DVDMS-SDT had no effect on the proliferation of normal HL-7702 liver cells.The effect of DVDMS-SDT on inhibiting the proliferation and inducing apoptosis of tumor cells was significantly better than that of Protoporphyrin IX（Pp IX）combined with ultrasound at the same concentration.DVDMS-SDT induced G2/M phase arrest in HCC cells,up-regulated the expression of G2/M phase marker proteins such as p21 and p27,and down-regulated the expression of cyclin-dependent kinases 1（CDK 1）and cyclin B1.Western blot analysis showed that DVDMS-SDT up-regulated the expression of Tumor protein p53（p53）and Bcl-2 associated X protein（Bax）,and down-regulated B cell lymphoma-2（Bcl-2）expression,which led to the activation of Caspase-3,ultimately initiated cell apoptosis.Based on in vitro results,the anti-tumor effects of DVDMS-SDT were further explored through nude mouse xenograft tumor models.The in vivo results showed that DVDMS-SDT significantly inhibited the growth of hepatocellular carcinoma xenograft,prolonged the survival time of model nude mice,and had no significant effect on the body weight.Immunohistochemistry showed that the expression of apoptosis-related proteins in the transplanted tumor was consistent with western-blot in vitro.These results suggest that DVDMS-SDT is a safe and effective treatment for HCC.Furthermore,this study examined the generation of reactive oxygen species（ROS）by fluorescence probs.DVDMS-SDT increased intracellular ROS and regulated the expression of apoptosis-related proteins through ROS.The addition of ROS scavenger N-acetylcysteine（NAC）could reverse DVDMS-SDT induced apoptosis and expression of apoptosis-related proteins.In addition,this study explored the types of ROS production induced by DVDMS-SDT in HCC cells.Firstly,cell viability experiments showed that singlet oxygen was not the main toxic medium produced by DVDMS-SDT.Secondly,the detection results of hydrogen peroxide（H₂O₂）and superoxide anion（.O₂^-）probes showed that hydrogen peroxide and superoxide anions were produced in the process of DVDMS-SDT,and were positively correlated with DVDMS concentration,ultrasonic intensity and ultrasonic radiation time.Under the same molar concentration（3μM）,the rates of hydrogen peroxide and superoxide anion production induced by DVDMS-SDT were much higher than Pp IX-SDT and Hematoporphyrin monomethyl ether（HMME）-SDT.In addition,hypoxia environment had no significant effect on apoptosis and hydrogen peroxide production induced by DVDMS-SDT.In summary,this study proved that DVDMS mediated SDT therapy can effectively inhibit the proliferation of HCC cells,induce cell apoptosis,and exhibit strong anti-tumor effect in vivo.The mechanism may be the regulation of p53/Caspase-3 pathway protein expression through the production of hydrogen peroxide and superoxide anions.Importantly,DVDMS-SDT could maintain high antitumor effect in low oxygen environment,suggesting that DVDMS-SDT may be more suitable for the treatment of tumor under hypoxia environment.