Correlation Between UCP2 And Melanoma And The Inhibitory Effect Of Celastrol Nanoparticles On Melanoma

Research BackgroundMelanoma is a malignant tumor derived from melanocytes with a high degree of sickness.The cause of the disease is not very clear.It may be because of pigment nevus,genetic factors,or UV radiation,the underlying molecular mechanism is not yet fully understood.Commonly used treatments include surgery,radiotherapy,chemotherapy,and immunotherapy,the results are far from being satisfactory.With the progress in cancer research,it has been found that the overexpression of mitochondrial uncoupling protein 2?UCP2?is tightly associated with the occurrence and development of tumors.However,the relationship between UCP2 expression and melanoma remains unclear.Celastrol?CEL?is a natural product derived from the root bark of Tripterygium wilfordii that has various biological activities.Previous studies have shown that celastrol can act on multiple molecular targets involved in tumorigenesis and development.However,the effect of celastrol on the expression of UCP2 protein in melanoma tissue is not clear.Besides,celastrol has poor water solubility,which usually leads to low bioavailability.A large dose of celastrol must be consumed to achieve optimum therapeutic effect.Higher doses of celastrol could cause adverse effects such as cardiotoxicity and hepatotoxicity.Therefore,it is particularly important to develop new drug delivery technologies to reduce severe adverse reactions and improve the efficacy of celastrol.Based on the aforementioned research background,we conducted the following research.Part One Expression and Significance of UCP2 in MelanomaPurposeDetection of UCP2 expression in melanoma tissues to explore its correlation with the clinicopathological characteristics of cutaneous melanoma aiming to identify new specific diagnostic and treatment targets for melanoma.MethodThe expression of UCP2 in 49 tissue samples of healthy skin,51 cases of compound nevus,and 65 cases of skin mucosal melanoma tissues was detected by immunohistochemical methods,and the correlation between UCP2 levels and clinicopathological characteristics of cutaneous melanoma was analyzed.ResultThe UCP2 positivity rates were low in normal skin tissue and compound nevus tissue,but relatively high in melanoma tissue?76.9%?.The expression of UCP2 protein in melanoma tissue was significantly higher than that in compound nevus and normal skin samples?P<0.05?.The expression of UCP2 in cutaneous melanoma tissues was not related to the patient's age,gender,lymphocyte infiltration degree,or presence or absence of ulcers?P>0.05?but related to tumor Clark grade and Breslow thickness?P<0.05?.ConclusionThe expression of UCP2 protein in melanoma tissues is significantly higher than that in normal skin and compound nevus.UCP2 expression level is related to tumor Clark grade and Breslow thickness.These data indicate that high expression of UCP2may influence the occurrence and development of melanoma.To some extent,it suggests that the tumor has a higher degree of malignancy and aggressiveness.Part Two Correlation and mechanism of UCP2 activity in melanomaPurposeInvestigation of the correlation and possible mechanism of inhibition of UCP2expression with melanoma biological behavior,oxidative stress,cell metabolism,chemotherapy resistance and its possible signaling pathways.Method1.UCP2 short hairpin RNA?shRNA?lentiviral plasmid expression vector and random control plasmid expression vector were used to transfect melanoma A375 cell line and melanoma SK-Mel-28 cell line.Stable UCP2 knockdown cells were constructed in each cell line.2.Effect of inhibition of UCP2 on the biological behavior of melanoma cellsThe effect of UCP2 inhibition on melanoma cell proliferation was detected by Inucyte Essen.The effect of UCP2 inhibition on melanoma cell migration and invasion ability was detected by Wound-healing cell scratch test and Transwell cell invasion experiment.The effect of inhibition of UCP2 on the tumorigenic ability of melanoma cells was detected by the three-dimensional spheroid growth assay.3.Inhibition of UCP2 on oxidative stress and cell metabolism in melanoma cellsThe effects of inhibition of UCP2 on mitochondrial membrane potential and levels of H₂O₂,ATP,lactate in melanoma cells were measured using specific kits.4.MTT assay was performed to detect inhibition of UCP2 on chemotherapy resistance of melanoma.5.The effect of inhibition of UCP2 on AKT/mTOR and MAPK/ERK signaling pathways was detected by western blotting.Result1.Two stable UCP2-knockdown clones?KD?and one control lentivirus-infected cells?LC?transfected with random control plasmid expression vector were established for each cell line and validated by western blotting.2.Impact of UCP2 inhibition on the biological behavior of melanoma cellsIn melanoma A375 cell line and SK-Mel-28 cell line,the proliferation rate of KD cells were significantly lower than that of LC cells?P<0.05?.In the Woundhealing assay for melanoma A375 cell line and SK-Mel-28 cell line,the widths of the scratches of KD cell clones were significantly larger than those of LC cells?P<0.05?.In the transwell assay for melanoma A375 cell line and SK-Mel-28 cell line,the numbers of invaded cells of the KD cell clones were significantly lower than those of LC cells?P<0.05?.In the three-dimensional spheroid growth assay,for the SK-Mel-28 cell line,the UCP2KD cells formed smaller spheroids compared with the control cell spheroids;for the A375 cell line,one UCP2 KD clone formed smaller spheroids compared with the control,while the other clone formed larger spheroids,but with markedly lower density based on fluorescence intensity analysis?P<0.05?.3.Effect of UCP2 inhibition on oxidative stress and cell metabolism in melanoma cellsIn melanoma A375 cell line and SK-Mel-28 cell line,the H₂O₂ levels,ATP levels and lactate levels of the two KD cell clones were lower than those of LC cells?P<0.05?.However,the mitochondrial membrane potential of the two KD cell clones were higher than that of LC cells?P<0.05?.4.Effect of inhibition of UCP2 on melanoma chemotherapy resistanceWith the increase of cisplatin concentration,the survival rate of KD cell clones decreased in A375 and SK-Mel-28 cells.After treating with cisplatin at concentrations of 1.67 and 5mM for 48 h,the UCP2 KD cells of both melanoma cell lines were more sensitive to cisplatin treatment compared with the LC cells?P<0.05?.5.Effect of inhibition of UCP2 on the signaling pathways in melanoma cells?1?AKT/mTOR pathwayIn melanoma A375 cell line and SK-Mel-28 cell line,the protein levels of phosphorylated Akt,phosphorylated p70S6K,and phosphorylated 4E-BP1 were significantly decreased in UCP2 KD cell clones compared of those of LC cells?P<0.05?.?2?MAPK/ERK pathwayIn melanoma A375 cell line and SK-Mel-28 cell line,the protein levels of phosphorylated ERK were significantly decreased in UCP2 KD cell clones compared of that of LC cells?P<0.05?.ConclusionUCP2 short hairpin RNA?shRNA?lentiviral plasmid expression vector effectively inhibits UCP2 expression.Inhibition of UCP2 decreases melanoma cell proliferation,migration,invasion,tumorigenicity,and leads to changes in the biological behavior of melanoma cells.Inhibition of UCP2 affects oxidative stress and cell metabolism of melanoma cells.Inhibition of UCP2 increases the sensitivity of melanoma to chemotherapeutic drug cisplatin.Furthermore,inhibition of UCP2 inactivated the Akt/mTOR and ERK pathways.UCP2 is suggested to be an effective target for the treatment of melanoma.Part Three Inhibitory effect of Celastrol nanoparticles on melanoma PurposeBy constructing novel celastrol-loaded nanoparticles?CEL-NPs?for the treatment of melanoma,the inhibition effect and safety of CEL-NPs in vivo and in vitro and its effect on the expression of UCP2 in mouse melanoma model tissues were studied,so as to provide a new method for clinical treatment of melanoma.Method1.Preparation of CEL-NPs using an“in situ drug conjugation-induced self-assembly”strategy.Verification of successful synthesis was performed by typical ¹H NMR spectroscopy and UV-Vis spectrometry,and calculate drug-loading content?DLC?and drug loading-efficiency?DLE?.Further validation of the expected effect was achieved by solubility investigation,serum stability test,particle morphology observation by transmission electron microscopy?TEM?,and mechanical radius measurement by dynamic light scattering?DLS?.High-performance liquid chromatography?HPLC?was used to determine drug release profile of CEL-NPs at different dosage ratios.2.In vitro experiments?1?CEL-NPs uptake by B16F10 mouse melanoma cells was detected by flow cytometry analysis?FCA?and confocal laser scanning microscopy?CLSM?.?2?The toxic effect of CEL-NPs,CEL,and methoxyl poly?ethylene glycol?-b-poly?L-lysine??mPEG-PLL?on B16F10 mouse melanoma cells and the safety of mPEG-PLL were assessed by MTT assay.3.In vivo experiment?1?By using a B16F10 mouse melanoma model,we studied inhibitory effects on tumors of different groups,which were separately treated with mPEG-PLL(22 mg kg^-1),free CEL(2 or 4 mg kg^-1),and CEL-NPs(containing 2 or 4 mg kg^-1 CEL).The alterations in mouse body weight were detected.?2?The in vivo distribution of CEL-NPs was studied by fluorescence imaging in vitro.?3?The antitumor effect of CEL-NPs and CEL was compared in vivo and the toxic response of main organs was studied by histological changes.?4?The effect of CEL-NPs and CEL on the expression of UCP2 protein in B16F10mouse melanoma animal tissue was determined by immunohistochemistry.Result1.Successfully assembled CEL-NPs have good solubility and stability.The drug loading content?DLC?and drug loading efficiency?DLE?of CEL in CEL-NPs were9.95 wt%and 53.9%,respectively.CEL-NPs have a spherical structure with a size of about 103.1±10.7 nm and hydrodynamic radius?Rh?of 65±6.4 nm.CEL-NPs showed a sustained slow in vitro drug release pattern.2.In vitro experiments?1?CEL-NPs can be effectively endocytosed by B16F10 cells as shown by FCA and CLSM.?2?The cytotoxicity of CEL-NPs was close to that of free CEL after incubation for24 h or 48 h.The IC₅₀ values for CEL-NPs and CEL at 48 hours were 2.81?M?1.26?g/mL?and 3.56?M?1.60?g/mL?,respectively.Anyway,mPEG-PLL showed negligible toxicities to mouse melanoma B16F10 cells.3.In vivo experiments?1?Tumor suppression test resultsThe tumor suppression rate of the low-dose CEL group was significantly higher than that of the mPEG-PLL group?P<0.001?.At the same dose,the tumor inhibition rate of the CEL-NPs group was higher than that of the CEL group?P<0.001?.The tumor suppression rate of the high-dose CEL-NPs?or CEL?group was better than that of the low-dose group?P<0.05 or P<0.001?.The body weight of the mice was monitored during the treatment.The average body weight of the CEL-NPs group was similar to that of the control group and the mPEG-PLL group,showing no statistical difference?p>0.05?.Compared with the CEL-NPs group,the average weight of the free CEL group was significantly reduced at the same dose?P<0.05?.?2?In vitro fluorescence imagingCEL-NPs-Cy5 was injected in the tumors of melanoma model mice.The fluorescence intensity at the tumor site stayed the same 3 h and 10 h after injection?P>0.05?.?3?Histopathological examinationThe area of necrosis was more extensive in the CEL-NPs treatment group.In the free CEL group,different degrees of degeneration and edema appeared in the liver and spleen.No ultrastructural changes in the organs were observed in the control group and in the CEL-NPs group.?4?Immunohistochemical resultsThe expression of UCP2 protein in the melanoma mouse model tissues of the high-dose CEL-NPs group and the high-dose CEL group was significantly lower than that of the control group?p<0.05?.ConclusionCEL-NPs were successfully synthesized,demonstrating good solubility,stability,and slow-release characteristics.These particles showed significant inhibitory effects on melanoma tumors both in vitro and in vivo which have the characteristic with high safety,low toxic and side effects.These particles can reduce the expression level of UCP2 protein in melanoma model tissues,providing a background for further clinical application.