| Mitomycin C (MMC) is a naturally occurring antibiotic produced by Streptomyces caespitosus that was first identified in 1956 and widely used in the clinic for the treatment of various human tumour diseases and has important activity in gastrointestinal, lung, liver, breast and cervical malignancies. However, the clinical application of MMC was limited by its toxic dose-related side effects, such as myelosuppression, thrombophlebitis, erythema and induration. So, it is important for clinical application of MMC to find out therapeutic methods to decrease adverse effect of drug and improve curative effect.Drug-protein binding is one of the essential factors in analyzing pharmacological mechanisms and pharmacodynamics of the drugs, because it can affect the transport, distribution, excretion, metabolism, and interaction with the target tissues. The most abundant protein in the human circulatory system is human serum albumin (HSA), which accounts for approximately 60% of the total protein corresponding to a concentration of 40 mg/ml (0.6 mmol L-1) in the blood. It can bind a wide variety of endogenous and exogenous compounds such as fatty acids, hormones, bilirubin, drugs, and diverse of metabolites. Thus, the investigation on the interaction of MMC with HSA is important for clarifying pharmacological of MMC. In the present paper, the binding of MMC to HSA have been investigated by fluorescence spectroscopy, UV-vis spectroscopy, circular dichroism (CD) spectroscopy and molecular docking methods. The fluorescence data showed that binding of MMC to HSA caused strong fluorescence quenching of proteins through a static quenching way, MMC with HAS form complex, and HSA had only one binding site for the drug. The binding constants of MMC to HSA at 288,298,308 K were3.35×104,2.71×104and 2.07×104 L mol-1, respectively. Thermodynamic analysis suggested that both hydrophobic interaction and hydrogen bonding played major roles in the binding of MMC to HSA. The binding distance between Trp in HSA and MMC is 4.55 nm. Thermodynamic analysis suggests that the binding process of MMC to HSA is spontaneous and exothermic reaction. The CD spectroscopy indicated that the secondary structureof the HSA was not changed in the presence of MMC. The study of molecular docking showed that MMC was located in the entrance of site I of HSA.Human hemoglobin (HHb), a major protein component in erythrocytes, not only carries O2 and CO2, but can reversibly bind with many kinds of endogenous and exogenous agents such as many drugs, herbicides, and insecticides. Since Hb is an important functional protein for reversible oxygen carrying and storage, the potential changes of conformation and function of HHb after binding of drugs have been a focus of study. The results of fluorescence spectroscopy showed that there are apparent interaction between MMC and HHb. The quenching of HHb by MMC is static quenching procedure from the formation of complex. The binding constants of MMC to HHb at 288,298,308 K were2.77×104,2.56×104and 2.15×104 L mol-1, respectively. Thermodynamic analysis suggests that the binding process of MMC to HHb is spontaneous and exothermic reaction and both hydrophobic interaction and hydrogen bonding played major roles in the binding of MMC to HHb. The result of synchronous fluorescence indicated that the polarity around the tyrosine residues in HHb decreased after binding. The CD spectroscopy indicated that the secondary structure of the HHb was not changed in the presence of MMC.The docking result showed that MMC located in the central cavity of HHb, partial to the subunitα2. Hyperthermia increases the efficacy of the treatment with several cytotoxic agent. Recently the result of a prospective randomized study were published, showing that thermo-chemotherapy appears to be more effective than standard chemotherapy. In the present paper, the effect of MMC versus MMC combined with hyperthermia on the cytotoxicity in HepG-2 cancer cell and the mechanism of the synergism were investigated. The result of MTT assay indicated that hyperthermia (43℃,1h)has a synergistic effect on the decrease of HepG-2 cell proliferation by MMC. The results also showed that thermotherapy can increase apoptosis of cancer cell induced by MMC and after hyperthermia at 43℃the expression level of NQO1 gene in the HepG-2 cell was increased, which suggest that those were one of the mechanisms of the synergism by MMC and hyperthermia.Cyclodextrin are cylic oligosaccharides composed of dextrose units joined through a 1-4 bond. Cyclodextrins with 6-8 dextrose units have been named a, P and y-cyclodextrin. The central cavity of these molecular is hydrophobic, while the rims of the surrounding walls are hydrophilic. This hydrophobic cavity forms inclusion complexes with a wide range of organic and inorganic guest moleculars, altering they physicochemical behhaviour, reducing their undesirable effects and decreasing adverse effect of drug. HP-CD(hydroxypropyl-β-CD), a hydroxypropyl derivative of P-CD, with improved water solubility properties and may be slightly more toxicologically benign, was considered a potential alternative ofβ-CD. In the present paper, the interaction of MMC with HP-CD, stability and biological activity of MMC—HP-CD complex have been investigated by competitive chromophore inclusion UV method, fluorescence spectroscopy, thermo-accelerating test and MTT method.The results of competitive UV spectroscopy showed that MMC with HP-CD form 1:1 complex and the binding constants of MMC to HP-CD at 298,308,318 K were611,468and 279 L mol-1, respectively. Thermodynamic analysis suggests that the binding process of MMC to HP-CD is spontaneous and exothermic reaction and van der Waals force played major roles in the binding of MMC to HP-CD. Thermo-accelerating test showed stability of MMC—HP-CD complex greater 2-fold than that of MMC. The results of fluorescence spectroscopy showed that quenching of HSA was decreased while HP-CD was added. The result of MTT showed cytotoxity of MMC—HP-CD complex on HepG-2 cell large than that of MMC. |
PDF Full Text Request