Hypericin-mediated Photodynamic Therapy Induces Apoptosis Or Necrosis In Mouse Myeloma Cell Line SP2/0

Photodynamic therapy(PDT) is a fast-increasing way for carcinoma treatment,and its advantage is effectively selective accumulation,non-invasiveness,little side effect and combination with other therapy for treatment of carcinoma.In clinical,PDT has been the fourth standard tumor therapy thereafter operation,radiotherapy and chemotherapy.However, with the restrain of photosensitizer and illuminant,PDT was only applied to minior volume or superficial carcinoma in clinical.Recently following the development laser and light canrrer,PDT as a beneficial tool for treatment major solid tumors oreven leukemia has been studied.Hypericin as second filial generation photosensitizer used in PDT,with it' s the powerful photosensiting,effectively selective accumulation in tumors and minimal cytotoxicity in the dark has been induction of clinical trials.The present study focused on exploring the correlationeffect of hypericin in vitro,and then the preliminary mechanism of apoptosis induced by hypericin in SP2/0 cells was investigated.To evaluate the cytotoxic effects of hypericin photoactived with five gradient illumination doses against seven cancer cell lines(including one cervical adenocarcinoma cell line Hela,three breast.carcinoma cell lines MDA-MB-435,MCF-7 and 4T1,one pancreatic.carcinoma cell line PANC-28,one myeloma cell line SP2/0 and one melanoma cell line B16) and human embryo kidney normal cell line 293T in Vitro.MTT method was employed to the cell viability assay clarified its potent cytotoxic effects on various cell lines. The results suggested that the high dose(10^-4M) for hypericin,in the dark,inhibited the growth of Hela cells,PANC-28 cells and SP2/0 cells respectively,but not on the others. Besides hypericin-photoactived significantly depressed the viability of the cells all above mentioned in a photo-dependent,drug-dependent and time-dependent way.The photokilling effects of hypericin-mediated PDT on the cells kept positive correlation.with drug dose or illumination dose.According to these results,we found that hypericin had strong inhibitory effect against those cell lines in vitro.To observe morphological changes in SP2/0 cells induced by photoactived hypericin at a dose of 11.28 J/cm² corresponding to 80-min illumination,inverted microscope,Hoechst 33342/PI,SEM and TEM were applied.It has been found that the lower dose(0.025μM or 0.05μM) for hypericin could induce typical apoptotic characteristics such as cell shrinkage, chromatin condensation and marginalization,nuclear fragment,mitochondria swell or vacuolization and the formation of apoptotic body,indicating the SP2/0 cell apoptosis;the higher dose(0.100μM) for hypericin could induce typical characteristics such as cellular swelling,membrane rupture the leakage of cell materials,implying the SP2/0 cell necrosis.In conclusion,photoactived hypericin could induce apoptosis or necrosis in SP2/0 cancer cellsTo assess apoptosis or necrosis inducing capability of hypericin-mediated PDT on SP2/0 cancer cells,DNA fragmentation,alkaline signal cell gel electrophoresis technique and PI staining-FACS analysis were used in the present study.The apoptosis induced by 0.025μM and 0.05μM of photoactived hypericin in dose-dependent manner was characteristised by clear DNA fragments and longer comet tails than these in control(p＜0.001),whereas 0.100μM of photoactived hypericin contributed to the large amounts of cell debris,only a few of serious apoptotic cells,and faint DNA fragments.The results indicated that photoactived hypericin possessed strong capability to induce apoptosis or necrosis in SP2/0 cells.By the assay of PI staining-FACS analysis,unexpectedly,we found that the cell cycle distribution failed to respond to 0μM,0.025μM,0.050μM and 0.100μM of photoactived hypericin.In fact,the result analysis cannot be taken by no typical DNA diploid peak showed in control.However,we found DNA content was decreased in a drug-dose dependent manner.It can be concluded that phot0actived hypericin induce apoptosis or necrosis.To elucidate the mechanism of apoptosis or necrosis induced by hypericin-mediated PDT on SP2/0 cancer cells,Western Blot assay was applied in our study.The results showed that photoactived hypericin in a lower dose partly induced the cleavage of PARP,otherwise, photoactived hypericin in a higher dose thoroughly induced the cleavage of PARR The Cleavage of PARP is dependent on caspases activity.At the time,it was found that the cleavage of Caspase-3 belonging,to Caspases family,which at the beginning and in the process of apoptosis,play a critical role in it,in a drug dose-dependent fasion.Taken together, the apoptosis or necrosis induced by photoactived hypericin,is related to the apoptotic signal conduction way for caspase-3-mediated-PARP。In conclusion,this study identifies that hypericin-mediated PDT.exerts significant cytotoxicity in vitro,the photokilling effect of which keep positive correlation with drug doses or illumination doses.Photoactived hypericin can induce apoptosis or necrosis of SP2/0.cancer cells in a drug-dose dependent manner.The apoptosis or necrosis induced by photoactived hypericin in SP2/0 cells might dePend on caspase-3-mediated-PARP way.