| Background The occurrence rate of cancer is rising and threatening the health of people. Many investigations show that the cancer is becoming the first killer. Controlling the cancer is now the strategy of hygiene of many countries and the comprehensive therapy of cancer is now the focus of the doctors in the world.Breast cancer is one of the most common cancer of women with a rising morbidity threatening the health of women. In Europe and America, breast cancer accounts for about 25～30% of women cancers. About 1.3 million people were diagnosed with breast cancer in the end of the 20th century and 400,000 people died from it. In our country, the breast cancer takes the second place in cities and fifth place in countries. Tumor treatment includs surgery, radiotherapy, chemotherapyand biological treatment as well as a variety of minimally invasive treatment, etc.Mini-invasive treatment includes photodynamic therapy, radiofrequency ablation, microwave, interventional therapyand so on.With the development of society, people tend to care more about the appearance, function and psychology than treating disease alone. PDT is playing a more and more important role in the treatment of cancer because of its advantages such as less trauma, less side effects, less cost, effectiveness, to protect the appearance、vital organ functionand so on.For precancerous lesions and early stage cancer, photodynamic therapy can cure them. And for the advanced cancer patients, PDT can be a palliative way to relieve the patients’pain. PDT can not only treat the superficial tumor, but also can treat the tumor of the respiratory tract, gastrointestinal tract, and bladder. Meanwhile, PDT could also play a role in non-tumor disease, such as age-related macular degeneration, pointed condyloma and resistance to bacteria and viruses. Hematoporphyrin Derivative(HPD) is the first generation of photosensitizer and proved to be effective after many years’ experimental researches and clinical trials, which is applied in clinic widely. PDT works by stimulating the photosensitizer accumulated in the cancer cells with laser of 630nm wavelength, producing singlet oxygen, oxygen free radical and thromboxane, causing the death of cancer cells and the tumor disintegration due to the embolism of microvessels. Compared with traditional therapies like surgery, chemotherapy and radiotherapy, PDT has many advantages:less trauma, less toxic, more selective, more adaptive, good repeatability, applied to palliative treatment, eliminating dormant neoplastic foci, protecting face and preserve the function of organs. In the process of PDT, the mechanisms of ROS production are divided into type I (redox reaction) and type II (energy transfer reaction). There are many species of type I, just like O2-,H2O2 and OH-,which are produced by the electron transfer after the interaction of photosensitizer and cellular content. The 1O2 from type II is the product of reaction of photosensitizer and ground state oxygen. Ros is an important reason to induce the death of cells. Researches have shown that PDT could cause the apoptosis and death of cancer cells.However, PDT has its shortcomings so that it is limited, just like the penetration depth of light. The tumor will relapse after PDT. One of the reasons is that the accumulation of photosensitizer in the cancer cells is limited. It is reported that calcitriol, namely,1,25(OH)2 vitamin D3, can inhibit cell proliferation of cancer cells. It can be combined with calcitriol receptor to adjust the proliferation and differentiation oftarget cells.Objective To understand the effect of calcitriol on the breast cancer cells, and select a proper concentration of calcitriol without affecting the growth of cells.To get to know the effect of calcitriolcombined with HPD-PDT on breast cancer cells and its mechanism. The ROS and apoptosis rate were detected. And the mRNA level of PpⅨ synthetase was detected. It also may provide theoretical proof for the clinical application of calcitriol combined with HPD-PDT.Materials and methodsCell culture The human cancer cell lines MCF7 and MDA-MB-231 were maintained in RPMI1640 medium with 10% fetal bovine serum,100 u/ml penicillin, and 100 u/ml streptomycin and incubated at 37℃ in a humidified atmosphere with 5% CO2.The detection of the concentration of calcitriol Breast cancer cells were cultured and seeded into 96-well plates at a density of 3×103 per well and incubated with concentration of 10-8M、10-10M、10-12M、10’14M、10-16M calcitriol for 48 hours. Then the viability was assessed via MTT assay. In comparison with blank group without calcitriol, proper concentration of calcitriol was chosen.Photodynamic treatment The cells were grouped into five groups, namely, experimental group (calcitriol+HPD+laser), HPD group (HPD+laser), calcitriol group (calcitriol+laser), laser group(laser alone), blank group. Breast cancer cells were seeded into 96-well plates at a density of 3×103 per well and incubated with calcitriol for 48 hours. Cells were then washed with PBS and serum-free RPMI 1640 medium containing HPD(10μg/ml) was added for further incubation with cells for 24 hours. Cells were then irradiated at a power density of 7.5J/cm 2 (XD-635AB; Xingda, Guilin, China). Immediately following irradiation, the medium was replaced with RPMI1640 medium supplemented with 10% FCS, and cells were incubated for 24 hours.Cell viability assayCell viability was examined by the MTT assay. Breast cancer cells were incubated withmedium containing MTT for 4 hours in the dark at 37℃. One hundred microliters DMSO was added into each well after removing the medium and incubated for 20 minutes. UV absorption was measured at 492 nm using a 96-well plate reader. Three independent experiments were conducted. The inhibition rate is calculated by the following formula.Inhibition Rate=(OD of blank group-OD of target group)/OD of blank group ×100%.Cell apoptosis analysis The apoptosis ratio was analyzed using Annexin V FITC Apoptosis Detection Kit (Jiancheng, Nanjing, China) according to the manufacturer’s instructions. Apoptotic cells were analyzed and quantified using FACScan flow cytometry (Becton Dickinson, USA). Tests were repeated in triplicate.Reactive oxygen species(ROS) detection The ROS value was detected by Multiscan Spectrum(Spectra Max M5, Molecular Devices, USA). Excitation wavelength was set to 480 nm, and emission was recorded at 510nm. It is conduted in the darkness in case the affection of light.Spectrofluorometric analysis The PpⅨ content of cells was assessed by Multiscan Spectrum(Spectra Max M5, Molecular Devices, USA). Excitation wavelength was set to 410 nm, and emission was recorded at 633 nm. An emission spectrum was recorded at the range of 600-750 nm. This fluorescence analysis was performed in the dark to minimize PpⅨ loss due to photobleaching.Real-time-PCR (RT-PCR)Total RNA of breast cancer cells was extracted with TRIzol (invitrogen) reagent according to the manufacturer’s instructions. First-strand cDNA was synthesized by using the high-capacity cDNA reverse transcription kit (Takara) with random primers. To examine changes in the level of mRNA for PBGD, UROS, CPOX Oand FECH, RT-PCR was performed on a ABI-7500. The primer pairs of these genes are listed in Table I. Parallel reactions were performed using primers toβ-actin as an internal control.Table I. RT-PCR primers to quantitatively measure the mRNAlevels of the PBGD, UROS, CPOX, and FECH genes.Statistical analysis Statistical analyses were performed using the SPSS19.0 software (SPSS Inc., Chicago, IL, USA). LSD t-test was applied when appropriate. A probability of< 0.05 was considered statistically significant. All values are expressed as mean±SEM from three different experiments.ResultsThe determination of concentration of calcitriol Figure 1 shows that the cytotoxic effect of a 48 hour-treatment with calcitriol is dose-dependent, and calcitriol at 10’12M was non-cytotoxic. Therefore,10"12 M calcitriol was considered optimal for use with breast cancer cells.The inhibition rate of cells after PDT The MCF7 cells’inhibition rate of experimental group, HPD group, calcitriol group, laser group are 89.03%±0.79%, 77.76%±2.27%,20.54%±2.32%,18.06%±3.60%, respectively. The MDA-MB-231 cells’inhibition rate of experimental group, HPD group, calcitriol group, laser group are75.82%±1.43%,46.38%±0.64%,16.98%±0.28%,13.49%±2.04%. The experimental group is highest among the four groups(P<0.01). There is no significant difference between calcitriol gourp and laser group(P>0.05). They are shown in Figure 2.The detection of Reactive oxygen species (ROS)30minutes after exposure, cells are collected to detect ROS. The MCF7 cells’ROS value of experimental group, HPD group, calcitriol group, laser group and blank group are 3.903±0.098, 2.713±0.218,2.239±0.945,1.835±0.127,0.561±0.004, respectively. The MDA-MB-231 cells’ROS value of experimental group, HPD group, calcitriol group, laser group and blank group are 3.628±0.045,2.269±0.175,1.649±0.296, 1.472±0.098,0.573±0.096, respectively. The experimental group is highest among the five groups(P<0.01). There is no significant difference between calcitriol gourp and laser group(P>0.05). The results are shown in Figure3.The detection of cell apoptosis The cells are collected 8 hours after light exposure. The MCF7 cells’rates of cell apoptosis of experimental group, HPD group, calcitriol group, laser group and blank group are 0.4407±0.01255,0.19±0.0094, 0.1023±0.0023,0.0680±0.02703,0.0047±0.0009, respectively. The MDA-MB-231 cells’rates of apoptosis of experimental group, HPD group, calcitriol group, laser group and blank group are 0.3110±0.0087,0.1980±0.0040,0.0903±0.0030, 0.0623±0.02630,0.0073±0.0009, respectively. The experimental group is highest among the five groups(P<0.01). There is no significant difference between calcitriol gourp and laser group(P>0.05). The results are shown in Figure4.The determination of fluorescence intensity The MCF7 cells’value of fluorescence intensity of experimental group, HPD group, calcitriol group, laser group and blank group are 386.056±13.540,285.871±13.292,2.702±0.199, 2.750±0.239,2.904±0.142, respectively. The value of MDA-MB-231 are 313.254±6.084,250.048±3.156,2.798±0.181,2.683±1.400, respectively. The fluorescence intensity of experimental group is highest(P<0.01). The pictures of fluorescence are shown in Figure 5.RT-PCR experiments Theporphyrin biosynthetic enzymes (PBGD, UROS, CPOX and FECH) are tested to investigate the mechanism. The results showed that only the expression level of copropopyrinogen(CPOX) oxidase was upregulated after calcitriol pre-treatment, while PBGD, UROS and FECH showed no changes in the mRNA level. The results are shown in Figure 6.In the current study, we demonstrated that 10"12M is a proper concentration of calcitriol via the MTT assay, which ensures that calcitriol will not affect the growth of breast cancer cells. After pretreatment of calcitriol, PDT was conducted. Next the viability was tested via MTT assay. The results showed that the viability in the experimental group was the lowest(P<0.01). The HPD group took the second place(P<0.05).There was no significant difference between calcitriol group and laser group. It showed that calcitriol could enhance the effect of HPD-PDT on breast cancer cells, while calcitriol cannot increase the death rate of cells alone. Then we continued the detection of ROS. The results told that the value of experimental group was the highest(P<0.01), meaning that in the experimental group, the production of ROS was highest, causing most death of breast cancer cells. And there was no difference between calcitriol group and laser group(P<0.05). The experiment of cell apoptosis detection indicated that the rate of cell apoptosis was the highest(P<0.01). Then we tested the fluorescence intensity of the five groups, showing that the value of experimental group was highest(P<0.05). In order to investigate the mechanism, we continue the RT-PCR to test the the expression levels of the porphyrin biosynthetic enzymes (PBGD, UROS, CPOX and FECH). Only the expression level of copropopyrinogen oxidase(CPOX) was increased after calcitriol pre-treatment, while PBGD, UROS and FECH showed no changes in the mRNA level. We conclude that calcitriol treatment can significantly increase CPOX expression at the mRNA level. Therefore, calcitriol may upregulate the CPOX expression to enhance HPD-induced PpⅨ level and fluorescence in breast cancer cells. CPOX is located at the mitochondrial outer membrane and plays a role in catalyzing coproporphyrinogen III to produce protoporphyrinogen. Previous studies have demonstrated that calcitriol or MTX increases the expression of CPOX, which results in a significant increase in the intracellular accumulation of ALA-induced PpIX in epithelial cancer cells and prostate cancer cells.We conclude that the induction of CPOX gene expression may play a role in the HPD-based fluorescence of tumor cells and the effect of photodynamic therapy.In summary, we have demonstrated for the first time that calcitriol can enhance the quality of HPD fluorescence imaging and can improve the efficacy of HPD-PDT by increasing PpⅨ levels in cells. Furthermore, we demonstrated that calcitriol not only can increase the level of HPD-induced PpⅨ, but it may also increase photosensitizer levels by elevating CPOX mRNA expression. Future studies are needed to investigate whether calcitriol can be effectively combined with HPD-induced in vivo and in the clinical setting. |
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