Pilot Study To Investigate The Effects And Mechanisms Of CAI On The Proliferation And Differentiation Of Keratinocytes

BackgroundPsoriasis is a common, recurrent and chronic inflammatory skin disease which, in most cases, lasts for lifetime, causing serious damage to the human healthy. Psoriasis is characterized by erythema, papules and scales with principal histopathologic changes including hyperproliferation of keratinocytes, infiltration of inflammatory cells, hemangiectasis in dermal papilla. The typical feature of psoriatic skin lesion is unique epidermal acanthosis and parakeratosis, which are caused by hyperproliferation and abnormal differentiation of keratinocytes. So far, the exact causes and mechanisms of psoriasis remain elusive; also there is no treatment with satisfactory therapeutic outcome in clinic. Classical drugs currently used in clinic to treat psoriasis cause lots of adverse reactions, leading to serious adverse reactions in patients after long-term use of these drugs. In recent years, although, the inhibitors of cytokines are demonstrated to be effective in treating psoriasis, the high cost, non-ignored side-effects and uncertainty of long-term administration restricts the use of cytokine inhibitors in clinic. Therefore, developing high effective and selective drugs with little toxic side effects and low cost will be very particularly important.Carboxyamidotriazole(CAI) is a synthesized small molecule which, in many studies, has been demonstrated to be a non-cytotoxic inhibitor of signal transduction via nonvoltage-gated Ca2+channels. It has been shown, in vitro and in vivo, that CAI inhibits metastasis and proliferation of various tumor cells, as well as angiogenesis. Previous data from our lab indicate that CAI has strong anti-chronic and-acute inflammation effects, and suppresses the generation of lots of pro-inflammatory cytokines such as tumor necrosis factor a (TNF-a) et al.From the above, we hypothesize that CAI might be a candidate of drugs used to treat psoriasis. Therefore, the overall goal of this project is to explore whether and how CAI executes its effects on proliferation and differentiation of keratinocytes by using biochemical and molecular biologic approaches, providing theoretical foundation to develop novel drugs to treat psoriasis.Methods1. Cell proliferation assay:Both typan blue dyeing cell-counting method and CCK-8method are employed to investigate the effects of CAI on the proliferation of human keratinocyte cell line (HaCaT) and human embryonic skin fibroblast cell line (ESF).The contribution of macrophages to HaCaT cell proliferation and the effect of CAI on cell proliferation is evaluated by using cell co-culture system of RAW264.7and HaCaT cells and CCK-8method.2. Cell apoptosis assays:Propium iodide staining for flow cytometry and phosphatidylserine exposure analysis are used to check whether CAI induces apoptosis and early apoptosis of HaCaT, respectively.3. Cell cycle distribution assay:Propium iodide staining for flow cytometry is used to observe the effects of CAI on HaCaT cell cycle distribution.4. Cell differentiation assay:RT-PCR and Western Blot are used to evaluate the mRNA levels (Involucrin, TGasel, Keratin10, Loricrin, Filaggrin, ANp63) and protein levels (TGasel and△Np63) of some biomarkers of cell differentiation in the presence or absence of CAI, respectively.5. Animal models:A common antipsoriasic drug screening model—mouse tail model, is used to check the effects of CAI on the generation of granular cells, obtaining preliminary data on whether CAI can improve psoriatic parakeratosis.A pilot study of adverse reactions to CAI is performed in the mouse tail model by obtaining general condition of mouse (weight, egestion and psychostic status).According to the requirements for skin irritation experiment in National Technical Standards of disinfection,2008, the irritation intensity of CAI on intact and damaged skin is measured to test if it is possible for CAI to be used as an external preparation to treat psoriasis.Results1. Cell proliferation1.1The data obtained by using typan blue staining cell-counting method indicate that CAI, at all doses (10,20,40μM) and all time points (24,48,72hour), significantly inhibited the proliferation of HaCaT cells in a dose-and time-dependent manner. Consistent with this, results with CCK-8method also showed all doses (2.5,5,10,20,40μM) of CAI suppress HaCaT cell proliferation at all time points (24,48,72hour) in time-and dose-dependent manner.1.2At low doses (2.5,5μM), CAI had no any effect on ESF cell proliferation after72hours treatment, however, at high doses (10,20,40μM), CAI significantly prohibited the proliferation of ESF cells. This is different from the effects of CAI on HaCaT cell proliferation, which showed that CAI, even at low doses (2.5,5,10μM), can substantially suppressed the proliferation of HaCaT cells, indicating the inhibitory effect of CAI at low concentration (2.5,2,10μM) is cell-type-selective. CAI at low concentration selectively inhibits the proliferation of HaCaT cells but not ESF cells.1.3RAW264.7cells treated with LPS alone for24hours experienced large morphologic change, extending pseudopodia, however, only a few of RAW264.7cells treated with CAI plus LPS extended pseudopodia. These results demonstrate that LPS alone was able to induce RAW264.7activation which was partly blocked by CAI.Neither LPS (1μg/mL) alone nor LPS-activated RAW264.7cells alone had any direct effect on HaCaT cell proliferation. Adding LPS into cell co-culture system of RAW264.7cells with HaCaT cells did not affect the proliferation of HaCaT cell either. Both LPS and LPS-induced RAW264.7cells did not influence the inhibitory effects of CAI on HaCaT cell proliferation.2. Cell apoptotic assayWe employed two methods to analyze the action of CAI at different concentration (5,20,40μM) for different time points (24,48hour) on cell apoptosis: propium iodide staining for flow cytometry and phosphatidylserine exposure analysis. The results derived from experiments with these two approaches were consistent, which showed that CAI only at high concentration (40μM) induced early apoptosis and late apoptosis of HaCaT cells.3. Cell cycle distribution assaysThe effects of CAI on cell cycle distribution of HaCaT cell was studied with method of propium iodide staining for flow cytometry. The data from these assays revealed that exposure to CAI significantly elevated the proportion of HaCaT cells in Go/G1phase, while substantially decreased the proportion of HaCaT cells in S phase. CAI at different time points (24,48,72hours) induced HaCaT G0/G1cell cycle arrest in dose-dependent manner.4. Cell differentiation assaysMessenger RNAs (mRNA) were prepared from untreated HaCaT cells or HaCaT cells treated with CAI (20μM) for12hours. RT-PCR was performed to check the mRNA levels of biomarkers of cell differentiation, including Involucrin, TGasel, Keratin10, Loricrin, Filaggrin and△Np63. The results of RT-PCR demonstrated that CAI significantly reduced mRNA levels of all molecules but filaggrin (P<0.05or0.01).The protein levels of TGasel and△Np63in HaCaT cells treated with DMSO or CAI (40μM) for48hours were detected with Western Blot. Densitometry analysis results demonstrated that CAI significantly decreased the protein level of TGasel (P<0.01), but not△Np63.The results indicated that CAI can regulate HaCaT cell differentiation and might be able to improve the abnormal cell differentiation and dyskeratosis.5. Animal model5.1With mouse tail model, we investigated the effect of CAI on the generation of granular cells in stratum granulosum. Pathological sections of epidermis were prepared from tails of mice, and were examined for the presence of a granular layer with an optical microscopy. We found that the epidermis from tails of mice treated with normal saline and PEG had a thinner or defect layer of stratum granulosum. On the contrary, the epidermis from tails of mice treated with methotrexate and high dose of CAI displayed an intact and thicker layer of stratum granulosum. These results demonstrated that both methotrexate and high dose of CAI were able to promote the generation of granular cells in stratum granulosum.Compared with normal saline, methotrexate administration significantly enhanced the percentage of scales with granular cells (10.00±1.49%for normal saline vs29.00±2.33%for methotrexate, n=12, P<0.001). Similarly, the percentage of scales with granular cells in the tails of mice treated with each dose of CAI (21.00±2.33%for30mg/kg,23.00±2.13%for40mg/kg) was much higher than that (12.00±2.00%) in tails of mice exposed to PEG400(n=12, P＜0.01), indicating that CAI was able to promote the generation of granular cells in mouse tail squamous epithelium, indicating that CAI can improve paraceratosis.5.2With mouse tail model, the general status of mice treated with CAI or vehicle control (PEG) was daily observed and recorded. Compared with PEG, CAI dose-dependently caused different degrees of apathetic and diarrhea sympotoms. Diarrhea symptoms in same individual eased after one week. Begun on the10th day after administration of CAI (40mg/kg), the weights of mice treated with CAI became significantly lower than that of mice treated with PEG. Together, these preliminary results verified that CAI could cause side effects of inhibition of central nervous system and gastrointestinal adverse reactions.Single-dose intact skin irritation test and repeated-dose intact skin irritation test (smearing CAI ointment at same site on the skin of guinea pigs once daily for14consecutive days) were practiced with homemade CAI ointment. The results showed that there was no erythema and edema on the skin of4guinea pigs; the skin irritation index of CAI ointment was0/4(4is the highest and the most irritable), and the irritation intensity level of CAI ointment was considered as non-irritation.In a single-dose damaged skin irritation test, erythema and edema were barely visible on the back skin of only1of4guinea pigs1hour after removing CAI ointment. The highest irritation index of CAI ointment was calculated as 0.5, and the irritation intensity level of CAI ointment was recognized as slight irritation. Topical application of CAI ointment is not irritable to intact skin, but causes slight irritation to damaged skin.Conclusions1. CAI significantly inhibits HaCaT cell proliferation, mainly through arresting HaCaT cells in G0/G1phase of the cell cycle and inducing HaCaT cell apoptosis. This inhibitory effect of CAI is cell-type-selective and is not modulated directly by macrophages.2. Based on the results which show that CAI is able to inhibit HaCaT cell proliferation, cause cell cycle arrest to prolong cell cycle progression, induce apoptosis, improve abnormal cell differentiation, and meliorate dyskeratosis, we speculate that CAI might have some potential application in treating psoriasis.