Effects Of TMPyP4-PDT On Human Cervical Cancer Cell Line Caski In Vitro

Background:Cervical cancer is one of the most common malignant carcinoma in women. Due to the widespread application of cervical cytology screening in recent decades, cervical cancer and precancerous lesions can be detected and treated earlier, and the morbidity and mortality of the advanced cervical cancer has been a marked decline. However, there is a younger trend in the incidence of cervical cancer in recent years.Photodynamic therapy(PDT) is a minimally invasive treatment for cervical cancer treatment. Meso-5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)(TMPyP4) is a kind of porphyrin derivatives and it is able to selectively gather in the tumor tissue, but quickly metabolized in normal tissues. So much attention is drawn in cancer treatment over a long period of time especially in photodynamic therapy aspect. There are some researches show that MCMs is a group of protein family and has been confirmed that it can be used as a specific marker of the proliferating cells. CA-Ⅸ is one of the isomers of carbonic anhydrase family and involves in cell proliferation and transformation. NF-κB is a nuclear transcription factor with multi-directional regulation. It is closely related to the tumor development,invasion and metastasis. P16is a cell cycle regulatory factor with negative regulation of cell proliferation and division. The aim of the research is to investigate the effects of TMPyP4-PDT on human cervical cancer cell line Caski in vitro and expressions of MCM2、CA-Ⅸ NF-κB and P16, and discuss the possible mechanism of action.Objective:To investigate the effects of TMPyP4-PDT on the survival of human cervical cancer cell line Caski and the expressions of MCM2、CA-Ⅸ、NF-κ B and P16, and discuss the possible mechanism of action. Methods:The cervical cancer Caski cells were divided into four groups:TMPyP4-PDT group, TMPyP4group, PDT group, and control group. We analyzed the effects of TMPyP4mediated photodynamic therapy(TMPyP4-PDT) on Caski by using MTT assay. Morphological changes of the cells were observed under an optical microscope using HE Staining Method. The cell apoptosis was observed using flow cytometry (FCM) with Annexin V/PI staining. The changes of the expressions of MCM2、CA-Ⅸ、NF-κ B and P16in Caski cells before and after TMPyP4-PDT were ananlyzed by using Fluorescent real-time quantitative PCR. Results:1. The suppressive effect of simple TMPyP4group gradually increased with the increase of the dosage(P<0.01)(3μM:8.6%,6μM:18.5%,15u M:32.2%,30μM:41.6%,60u M:48%)in the same conditions of the laser energy density. The suppressive effect of simple PDT group was not obvious(P>0.05). The suppressive effect of TMPyP4-PDT group increased with the increase of the dosage and the light energy density(P<0.01).2. More adherent cells without nuclear pyknosis and nuclear fragmentation of simple PDT group were observed under the light microscope; cytoplasmic vacuolization, cell shrinkage, nuclear fragmentation and apoptotic body formation of simple TMPyP4group and TMPyP4-PDT group could be observed under the light microscope.3. FCM analysis showed that the apoptosis rates of Caski cells were (5.67+0.35)%,(13.50±0.62)%,(22.83±2.35)%,(34.47±1.88)%and (46.43±1.46)%when the laser energy was4J/cm2and the doses of TMPyP4were3,6,15,30,and60u M and the treatment time was4h.4. The expressions of MCM2, CA-IX and NF-κB mRNA were decreased significantly at4h after3,6,15,30and60μM TMPyP4compared with normal control group(P<0.01) under the conditions of the light energy density of4J/cm2and the expressions of P16mRNA were increased significantly compared with normal control group(P<0.01).Conclusions:TMPyP4-PDT have distinctive inhibition effects on cervical cancer cell line Caski and can induce the apoptosis of Caski. The apoptosis gradually increased with the increase of the concentration of TMPyP4. After effects on Caski cells for a period of time, TMPyP4-PDT may inhibit the proliferation and metastasis of tumors through up-regulating the expression of P16and down-regulating the expressions of MCM2, CA-Ⅸ and NF-κB.