Inhibitory Effect Of Ultrasound With Hematoporphyrin On Mouse Hepatoma H22 Cells

Tumor, especially malignant tumor, is a direct threat to the safetyof human life. In 1996, there were 10 million new tumor cases of worldwidescope. Meanwhile 160-200 million new tumor cases occurred each year inour country. Experts have forecasted that the incidence rate could bedoubled in the next 20 years, therefore, the control and prevention of tumorsare the most important medical research in the world. Currently, there arelots of conventional treatments for tumor as surgery, radiation therapy andchemotherapy. However, these methods all have their pros and cons:surgery, surgical excision of tumor, is better in early treatment butadvanced ineffective; radiation therapy is failed on widely spread or partialtumor invasion, and it also brings some near or long-term side effects;Chemotherapy is the main cancer treatment using chemical means ofantineoplastic agents, which have strong side effects, mainly includingnausea, vomiting, alopecia, leukopenia losing, and so on. Hence, searchingfor a new method of cancer prevention with good selectivity and littleside-effect is a medical hot. Ultrasound, as a form of mechanical energy,has a wide range of uses in the fields of medicine and biology, particularlyin the treatment of cancer, what attracted widespread attention at home andabroad. In 1989, Umemura S et al first reported that there was anti-tumoreffect of hematoporphyrin(Hp) activated by ultrasound, so calledsonodynamic therapy. Generally speaking, sonodynamic therapy is based onthe ultrasound that can penetrate deeply into biological tissue and activateHp accumulated in tumor, which produced lots of singlet oxygen and otherfree radicals, causing irreversible damages to tumor cells. Because ofpossessing excellent pertinency, penetration ability and no wound fornormal tissues, the therapy has advantages for curing deep cancers, which istaken attention of cancer research experts all over the world. Currently,synergistic effects of ultrasound and hematoporphyrin on tumors were stillin the stage of research. The results achieved as follows: The radiation source used: plat and focused ultrasound; sonosensitizerused, porphyrin agents such as hematoporphyrin, porphyrinⅡand ATX-70,and non-porphyrin agents, such as anti-cancer drugs and chemotherapydrugs; parameter reference used, 210 kHZ-2.0 MHZ frequency and 150mW/cm~2-3.2 W/cm~2 intensity, 15-60 s treated time intervel in vitro and 2-10min in vivo; tumor cells used, S180, AH130, HL-60, VX2. LiBr and humantumor cells, K562 and so on; related mechanism achieved, cavatition,singlet oxygen, hydroxide radicals alkyl oxygen radicals and so on.In this dissertation, 1.43 MHz, 2 W/cm~2 ultrasound is used to activate100μg/ml hemaporphyrin in order to study the inhibitory effect on H22 cellsin vitro and in vivo on the backgroud of international research developmentand our previous results and this research is supported by national naturalscience foundation of china. The results obtained are as follows:(1) Hp absorption at different concentrations: The results showed thatthere exsited saturated situation, that was, the concentrations in H22 cellswas not always raised as incubation time increased. At different Hpconcentration, the time interval relative saturation point reached is different.However, the H22 cells absorbed Hp at relative saturation point after 60minutes, what was the best incubation time interval before ultrasoundtreatment.(2) Treatment parameter selection: The results showed that thereexsited threshold of ultrasound intensity and Hp concentration. Underultrasound treatment alone, there was no significant effect on cell survivalif the sound intensity is less than 2W/cm~2. However, ultrasound hadobviously inhibitory effect on cell survival, even above 50%, while hesound intensity is more than 2 W/cm~2; Under ultrasound combined with Hptreatment, there was no significant synergistic effect on cell survival if Hpconcentration below 50μg/ml, while above 50μg/ml, it was oposite situation.Different parameter role in tumor cells could lead to the destruction ofdifferent mechanisms, taking into account the purpose of this study, wechoose the optimized treatment of 2W/cm2 100μg/ml, 60s to study thedynamic physiological and biochemical changes and explore the mechanismof cell death. (3) Physiological and biochemical analysis: after designed treatment,we found there were significant changes of apoptosis-relative proteins,including Caspase-3, Fas/FasL, and Bcl-2, suggested that the death processmight also exist in both endogenous and exogenous apoptosis pathway.(4) Inhibitory effect on H22 in vivo: after reconstruction of H22 solidtumor, we found that ultrasound treatment alone revealed a weak inhibition,compared with the control group significantly. However, UH group, haveextremely significant anti-tumor effect, weight and size of inhibition rateswere 46.3% and 60.8%.