The Antitumor Effect And Mechanism Of Hematoporphyrin Monomethyl Ether Mediated Photodynamic Therapy On Bone And Soft Tissue Sarcoma

Objectives: To investigate the effects and mechanisms of hematoporphyrinmonomethyl ether mediated photodynamic therapy (HMME-PDT) on sarcoma.Methods:1. In vitro, The intracellular uptake of HMME on osteosarcoma(OS) cells(LM8) and myoblast cells (C2C12) incubated at various time points andconcentrations of HMME by flow cytometry method. The optimal incubation time ofHMME and the bone and soft sarcoma cells viability was measured by3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Theapoptosis of OS cells were detected by flow cytometry method with AnnexinV-FITC/PI staining. The apoptosis of cells was further observed by Hoechst33342staining. The expression levels of caspase-3, Bcl-x, p53and RelA mRNA weredetected by real-time quantitative reverse transcription-polymerase chain reaction(QRT-PCR). The relative expression levels of caspase-1,3,6,9and PARP weredetected by Western blotting. The irreversible inhibitor of caspase (Z-VAD-FMK) andnecrosis (Nec-1) was performed to detect the type of PDT-mediated cell death.2. Invivo. The OS model of mouse was established. The efficacy of antitumor wasevaluated by the size and the weight and Hematoxylin eosin (HE) staining was usedto observe the histological changes of tumor. The relative expression levels ofcaspase-1,3,6,9and PARP were detected by Immunohistochemistry (IHC).Results:1. The intracellular uptake of HMME was time-and dose-dependent andHMME can selectively accumulate in OS cells while normal cells absorbed much less.4hours was considered to be the optimal incubation time. HMME-PDT can markedlykill the sarcoma cells and the killing effect increased with HMME concentration andenergy intensity. The apoptosis rates in HMME-PDT groups significantly increasedboth adherent and suspension cells. It was observed typical changes, such askaryopyknosis, cell rounded and so on, by Hoechst33342stained. the caspase-3mRNA and relative protein expression were markedly up-regulated. However, theBcl-x, p53and RelA mRNA and relative proteins expression were significantly down-regulated. The results showed that apoptosis was the major form of cells deathand might be closely with caspase-dependent apoptosis after HMME-PDT.2.Comparing with the blank group, the tumor volume and the tumor weight weremarkedly decreased. The widespread necrotic areas were observed by HE in tumor.The expression levels of caspase-1,3,6,9and PARP were significantly up-regulatedin the HMME-PDT groups.Conclusions: HMME can selectively accumulate in OS cells while normal cellsabsorbed much less. HMME-PDT showed potent killing effect, which was time-anddose-dependent on OS cell. The apoptosis was the main type of HMME-PDTmediated cell death and HMME-PDT significantly inhibited the tumor growth in vivo,which are closely correlated with caspase-dependent pathway.