Preliminary Study On Mechanisms Of Hematoporphyrin Monomethyl Ether-Photodynamic Therapy Induced Hela Cells Death

Objective Hematoporphyrin Monomethyl Ether (HMME) is a novel porphyrin-related photosensitizer, which has higher selective uptake of tumor tissue, stronger photodynamic effect, lower toxicity and shorter-term skin photosensitizations than the first generation of photosensitizers, and is a promising photosensitizer for photodynamic therapy(PDT). However, the mechanisms of HMME-PDT induced target cell death have not been understood. So, a preliminary study was undertaken for the purpose of understanding the mechanisms of HMME-PDT induced target cell death. Methods and Results (1) Effect of different reactive oxygen species on HeLa cells death mediated by HMME-PDT: The HeLa cell were treated by HMME-PDT, The rate of apoptotic or necrotic cells was respectively detected on flow cytometry through double staining of Annexin-V and popodium iodide afte PDT 6 or 24 hours. Results showed that HMME-PDT could induced HeLa cell death through necrosis and apoptosis. hi an early phase after PDT, Sodium azide(the singlet oxygen quencher) could protected HeLa cell from necrosis induced by HMME-PDT, whereas D-Mannitol (the hydroxyl radical quencher) protected HeLa cell from apoptosis also induced by HMME-PDT. (2) Damages of endoplasmic reticulum and mitochondria by HMME-PDT and their roles in results of the HeLa cells death: The [Ca2+]j was measured with fluorescence dye Fura-2/AM by a fluorospectro-photometry. The cytochrome C translocation and Sarco/endoplasmic. reticulum calcium-ATPase2 (SERCA2) protein degradation in PDT treated HeLa cells were analyzed by Western blotting. The activity of caspase-3 was detected with the ApoAlert?Caspase-3 fluorescent assay kit. The cell survival rates and the percentages of apoptotic or necrotic cells were respectively analyzed on MTT asssay or flow cytometry through double staining of Annexin V and popodium iodide(PI). Results revealed that HMME-PDT immediately induced[Ca2+]i elevation in a PDT dose-dependent manner, which could inhibited by sodium azide or D-mannitol. [Ca2+]i elevation after HMME-PDT declined by BAPTA/AM (an intracellular calcium chelator) could rescued HeLa cells from apoptosis and necrosis results to increase cell survival. HMME-PDT was also caused SERCA2 protein degradation and cytochrome C translocation from mitochondria to cytosol in a PDT dose-dependent manner, which could inhibited by sodium azide, D-mannitol or BAPTA/AM. Caspase-3 activated by HMME-PDT could inhibited by sodium azide, D-mannitol or BAPTA/AM, too. (3) Role of Extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38MAP kinas in HMME-PDT induced HeLa cells death: The total and phosphorylated ERK1/2 or pSSMAPK were dectected by Western bolting. The cell survival rates and the percentages of apoptotic or necrotic cells were also respectively analyzed on MTT asssay or flow cytometry through double staining of Annexin V and popodium iodide(PI). Results demonstrated that ERK1/2 and p38MAPK activity were induced in HeLa cells after HMME-PDT. When HMME-PDT treatment blocking of the ERK1/2 or p38MAPK activity with PD98059(a special inhibitor of ERK1/2) or SB203580 (a special inhibitor of ERK1/2), significantly increased HeLa cells. apoptosis and necrosis results to decrease cell survival.Conclusions Reactive oxygen specieses formation in HeLa cells after HMME-PDT may be through two ways, Type I and Type II photodynamic reactions. Singlet oxygen generated by Type II photodynamic reaction mainly induce necrosis in HeLa cells, but hydroxyl radical generated by Type I photodynamic reaction mainly induce apoptosis. HMME-PDT can damage HeLa cells endoplasmic reticulums and mitochondria results to inducing apoptosis and necrosis, but HeLa cells can activate ERK1/2 and p38MAPK to protect from apoptosis and necrosis.