Mechanism Of The Effect Of Hexyl-aminolevulinate Photodynamic Therapy Combined With Calcitriol On Human Skin Squamous Cell Carcinoma A431 Cells

BackgroundCutaneous squamous cell carcinoma(CSCC)is the most common human cancer in the world,and its incidence is increasing.Surgical resection is a common method for the treatment of squamous cell carcinoma.However,special sites,scars,elderly patients can not tolerate surgery or extended resection are some of the disadvantages of surgical therapy.In the past few decades,as a non-invasive treatment,Photodynamic therapy(PDT)has become a choice for the treatment of some non-melanoma skin cancer in China.Aminolevulinate(ALA)is the most widely used photosensitizer in clinic at present.It is absorbed by abnormal proliferating cells and produces protoporphyrinⅨ(PpⅨ)in mitochondria,which produces reactive oxygen free radicals(ROS)under specific wavelength of light excitation,resulting in toxicity.However,the stability of ALA is not good,and the permeability to the biological barrier is poor,so it is difficult for the deeper tumor tissue ALA to enter effectively,resulting in ALA-PDT can not be significantly effective.Hexyl-aminolevulinate(HLA)is an effective photosensitizer.Hexyl group is added to ALA to make it more stable,lipophilic and easier to penetrate the biological barrier,thus reaching deeper skin tissue and producing more PpIX than ALA,thus enhancing the efficiency of photodynamic.Although Photodynamic therapy has its advantages,it also has limitations in terms of indications.The main reason is that it plays a very importantrol e in invading the tumor tissue in the deep layer of skin tissue,and it is ther oot cause of postoperative recurrence.Some studies have shown that calcitriol can change the expression of key enzymes in the heme synthesis pathway of exogenous photosensitizer ALA,resulting in the production of more PpIX,but whether calcitriol has a synergistic effect on HLA,thus further enhancing th e photodynamic effect,has not been seen.ObjectiveTo study hexyl-aminolevulinate photodynamic therapy combined with calcitriol on human skin squamous cell carcinoma in vitro,so as to provide reference basis for improving the photodynamic effect of skin deep in tumor in the future.Methods1.The fluorescence intensity of PpⅨ was detected after A431 cells were incubated with HLA.The inhibitory effects of calcitriol,HLA-PDT and combination group on human squamous cell carcinoma A431 cells were detected by CCK8.Drug combination index CI(combinationindex)was introduced to evaluate whether the combination group was synergistic and its intensity,and to determine the parameters of the follow-up experiment.2.The effects of ALA,HLA and calcitriol on A431 cells were compared by observing cell morphology,cell scratch test,cell death staining,ROS production and the changes of mitochondrial membrane potential.3.The cell cycle detection,the mode of cell death,the key enzymes of porphyrin synthase system and the mRNA expression level of apoptosis-related proteins were detected after combined treatment,in order to further explore the possible ways of combined therapy to play a role.Results1.The cell culture medium containing HLA had no obvious toxicity to A431 cells when the concentration was less than 200 μM,and calcitriol solution(0.01nM～100nM)at low concentration had no obvious effect on the proliferation of A431 cells,and the inhibition rate of A431 cells was enhanced with the increase of drug concentration and incubation time when the concentration was 1000nM.The inhibitory effect of HLA-PDT on A431 cells was dependent on HLA concentration and light intensity.Compared with the single treatment group,the inhibitory effect of Comb group was stronger.When the half inhibition effect was exerted,the calcitriol concentration was 83.5 μM,and the light energy was 2.4J,which showed a strong synergistic effect.2.Compared with ALA,HLA-PDT has stronger inhibitory and killing effect on cells,higher level of ROS and lower level of MMP.Calcitriol combined with calcitriol can further enhance the effect of ALA-PDT and HLA-PDT.3.After combined treatment,more cells were blocked in G1 phase of G0 shock,compared with the single treatment group,more apoptosis,up-regulated mRNA expression of porphyrin synthase CPOX,down-regulated FECH expression and up-regulated mRNA expression of apoptosis-related protein Bax.Calcitriol combined with HLA-PDT induced cell death through apoptosis.