The Anti-cancer Effect And Mechanism Of Photodynamic Therapy Mediated By LS-HB On Malignant Melanoma

Background:Malignant melanoma is a serious threat to human health.In addition to the effective effect of surgical resection for early stage tumor,the advanced melanoma is often resistant to radiotherapy and chemotherapy.Photodynamic therapy(PDT),as a novel tumor treatment technology,has been widely used in a variety of solid tumors.Preliminary studies indicate that PDT may also be an effective treatment strategy for malignant melanoma.However,there are some problems in clinical photosensitizers,such as low purity,high dark toxicity,poor water solubility and poor tissue penetration of matching laser,so it is urgent to seek new photosensitizers with high efficiency and low toxicity.LS-HB is a new type of chlorin e6 photosensitizer,which is a stable product obtained by chlorophyll transformation.It has the advantages of simple synthesis,single composition,strong photosensitizing power and easily soluble in water,so it is of great significance to explore its photodynamic anti-tumor effect and molecular mechanism.Purpose:This study was aim to clarify the physical and chemical properties of LS-HB and explore the anti-tumor effect and mechanism of LS-HB PDT on malignant melanoma in vivo and in vitro,so as to provide experimental basis for the drug development and clinical application of LS-HB.Methods:The physicochemical properties and singlet oxygen output of LS-HB were detected by spectrometry,photobleaching experiment and SOSG oxidation experiment.CCK-8 was used to test the inhibition of LS-HB PDT on malignant melanoma B16F10 and A375 cells and its influencing factors.The cytotoxic mechanism of LS-HB PDT on B16F10 cells was investigated by using reactive oxygen species probe DCFH-DA,mitochondria-specific probe MTG,mitochondrial membrane potential probe JC-1,apoptosis dye Annexinv FITC/PI combined with laser confocal microscopy,flow cytometry and Western blot.The anti-tumor effect of LS-HB PDT on malignant melanoma in vivo and its possible mechanism were explored through the tumor inhibition experiment in mice.Results:Results:The longest absorption wavelength of LS-HB in the visible light range was 660 nm,the photobleaching rate was slow,and the singlet oxygen output increased with the increase of light dose and LS-HB concentration.The results of in vitro test showed that LS-HB(0-32 μg/mL)had no significant dark toxicity to B16F10 and A3 75 cells.It is found that the light dose had a good dose-effect relationship in the range of 0-10 J/cm2,and the inhibitory effect tends to be saturated when it reaches 20 J/cm2.And LS-HB PDT could induce B16F10 cells to produce reactive oxygen species.It was mainly distributed around the nucleus in the cell,and was more consistent with the distribution of mitochondria.The mitochondrial membrane potential decreased significantly after light exposure.The expression of caspase-9,caspase-3 and PARP decreased while the expression of its hydrolysate increased in a concentration-dependent manner.Under the microscope,it could be seen that the cell shape shrank,rounded,and even collapsed.Flow cytometry showed that as the dose of LS-HB increased,the rate of apoptosis and necrosis increased.The results of in vivo tests showed that the tumor growth in the LS-HB PDT treatment group was significantly slowed,and the inhibition rates in the 1 mg/kg and 2 mg/kg treatment groups were 58%and 83%,respectively.Tumor cells were obviously necrotic,blood vessels were congested or thrombosis is formed,but there was no obvious change in normal organs and tissues.Conclusion:The best excitation wavelength of LS-HB was 660 nm.It had good light stability,high singlet oxygen output,and showed good second-generation photosensitizer characteristics.LS-HB had obvious photodynamic inhibitory effect on malignant melanoma B16F10 and A375 cells after 660 nm light irradiation.The main mechanism was to induce cell apoptosis through the mitochondrial-mediated caspase pathway at low doses,and directly induce cell necrosis at high doses.LS-HB PDT had obvious anti-tumor effect on B16F10 xenograft tumor models,and its mechanism might be related to the damage of tumor cells and blood vessels in tumor tissues.