Mice S180, L1210 Tumor Cell Cycle And Its Mechanism Of Exogenous Pp ¢ù

In recent years, two relatively new approaches that named "Photodynamic therapy (PDT)"and "Sonodynamic therapy (SDT)" were proposed for cancer treatment, they are based on the synergistic effects of visible light (or ultrasound) and photosensitizer (sonosensitizer) which means the photosensitizer (sonosensitizer) can be activited by visible light (or ultrasound) to perform the tumor cell growth inhibition. As the core composition of PDT (SDT), photosensitizer (sonosensitizer) plays a very important role. So a series of photosensitizers were developmented by the related scholars in home and abroad to optimize the efficacy of PDT (SDT). As so far, porphyrins are the relatively commom used photosensitizers (sonosensitizers). Protoporphyrin IX (PpIX) is an effective compound of hematoporphyrin derivatives, as a relatively ideal photosensitizer (sonosensitizer), it has been studied carefully in PDT, but there are few reports about PpIX in SDT and the cell killing effect by ultrasound activating PpIX is still on the primary study. The mechnism that PpIX act on the tumor cells are also unknown.On the background of international research development and our previous results, this paper is supported by the National Natural Science Foundation of China (Grant No.39870240 and No. 30270383). The S180, L1210 cells were used for experiment model to study the the cell killing effect of exogenous PpⅨat different concentrations and disscussed the action mechnism of exogenous PpⅨ. The experimental conclusions are as follows:(1) The different cell growth inhibition effects were shown when cells tratead with PpⅨat different concentrations:In S180 and L1210 cells, compared to control, it has no growth inhibition when PpIX at the concentration of 0.1,0.5μg/ml, a slightly cytotoxicity of PpⅨwas showed at 1μg/ml (p>0.05) and significant loss of viability was detected at 5,10,20, and 40μg/ml PpⅨ(p<0.05) in a dose-dependent manner. In addition, the cell viability decreased gradually, reached its minimum at 8 h and increased from 8 h to 72 h.(2) The cell cycle was analyzed with Flow cytometric when cells tratead with PpIX at different concentrations for different time, and the results showed that:when S180, L1210 cells treated with PpⅨat different concentrations for 12 h, compared to control, the percent of S-phase cells were increased as the PpⅨconcentrtions increased, when concentration of PpⅨwas at 40μg/ml, the percent of S-phase cells was increased from 42.65%(control) to 58.21% in S180 cells and from 42.89%to 55.37% in L1210 cells; when S180, L1210cells treated with PpⅨat different concentrations for 24 h, compared to control, the percent of S-phase cells were also increased as the PpⅨconcentrtions increased, when concentration of PpⅨwas at 40μg/ml, the percent of S-phase cells was increased from 31.62%(control) to 52.59%in S180 cells and from 36.87% to 49.48% in L1210 cells; when S180, L1210 cells treated with PpⅨat different concentrations for 36 h and 48 h, compared to control, there was no change in percent of S-phase cells as the PpⅨconcentrtions increased. The results suggested that:PpⅨcan inhibit the growth of S180, L1210 cells, which may to relate to induce the S-phase arrest.(3) The protein expression of cell cycle-related molecule was detected by Western-Blot when cells treated with PpⅨat various concentrations. The results indicated that:when cells treated with the PpⅨat various concentrations for 12,24,36,48 h, the protein expression of CDK2 have no change compared to control as PpⅨconcentration increased; compared to control, the protein expression of cyclinA have no change as PpⅨconcentration increased when cells treated with PpⅨfor 12,36,48 h; when cells treated with PpⅨfor 24 h, the protein expression of cyclinA, p53, p21 increased as PpⅨconcentration increased. In L1210 cells, compared to control, the protein expressions of CDK2, cyclinA have no change when cells treated with PpⅨfor 12,24,36 h; the protein expression of p53 have no change, the protein expression of p21 increased slightly when cells treated with PpⅨfor 24 h as PpⅨconcentration increased.(4) The important tumor suppressor gene p53 and p21 play a significant role in the process of S phase arrest induced by PpⅨ. The possible reason is that DNA damage induced p53 protein expression, further upregulate p21, as a broad inhibitor of CDK, p21 inhibited the activity of CDK2 and slowed down DNA replication. In L1210 cells, the tumor suppressor gene p53 didn't work in the process of S phase arrest. But whether the p21 was upregulation via p53-independent manner? The exact mechnism need to investigate further.(5) The mRNA expression of cell cycle-related molecule was detected by RT-PCR. When cells treated with PpⅨat various concentrations, the results showed that:The mRNA expressions of CDK2, cyclinA, cyclinD1, cyclinE, p53, p21 have no change when cells treated with PpⅨfor 12 h as PpⅨconcentration increased in S180, L1210 cells, which represent that PpⅨinfluecse possibly the mRNA translation of cell cycle-related molecule but not gene transcription, for example PpⅨmake frequency of mRNA translation rise or mRNA stability increase. Eventually lead to upregulate the protein expression. But the exact mechnism need to investigate further.