Anticancer Activity Of Thymic Immunosuppressive Pentapeptide (TIPP)

ObjectivesCancer is a group of diseases involving abnormal and uncontrolled cell growth and the potential of penetration and diffusion to other parts of the body.Cancer is considered as the leading cause of death in the world.In 2008,about 12.7 million cancer cases and 7.6 million cancer deaths occurred in the world,whereas in 2012,approximated 14.1 million new cancer cases and 8.2 million cancer deaths occurred globally.Therefore,the study of antitumor drugs with high efficiency and low toxicity has been the focus of drug research.Our laboratory began to study thymic immunosuppressive extract(TISE)in the 1980s.It was found that TISE could inhibit lymphocyte proliferation,immune and allergic responses effectively both in vitro and in vivo.TISE effectively suppressed delayed hypersensitivity reaction in mice and passive cutaneous anaphylaxis in rats,significantly extended the survival time of transplanted skin in rats,and apparently inhibited OVA-induced asthma attacks in guinea pigs.For further investigation of the immunosuppressive activity and mechanism,TISE derived from calf thymus was digested by micrococcal nuclease and separated by RP-HPLC and a novel pentapeptide with the sequence of Ala-Glu-Trp-Cys-Pro(MW=604.68)was obtained and named as thymic immunosuppressive pentapeptide(TIPP).Lian Q et al.proved that TIPP could effectively suppress the allergic and inflammatory responses in allergic mice via blocking MAP kinases/NF-KB signalling pathway.Lian Q et al.also demonstrated that TIPP efficiently inhibited the IgE-mediated activation of RBL-2H3 cells through targeting MEK/ERK and NF-κB signaling cascades.On the basis of the broad pharmacological effects of TIPP,we hypothesized that TIPP may also have a role as anticancer agent.So we decided to investigate its anticancer activity.This was the first time to study the anticancer activity of TIPP.To confirm this supposition,we first studied the anticancer activity of TIPP on cellular level and evaluated the effect of TIPP on the animal tumor models normal organs.The results of this project may provide a new way for cancer treatment and lay foundation for the deep research and development of TIPP.Methods1 Study on the anticancer activity of TIPP in vitroThe cytotoxicity and inhibitory activity of TIPP on the proliferation of breast cancer cell line MCF-7 and chronic myeloid leukemia(CML)cell line K562 were evaluated.Breast cancer and CML cancer cells were cultured and seeded in 96 well plates,and were treated with five different concentrations of TIPP for 48 h.Subsequently,20 μl aliquots of 5 mg/ml MTT(Sigma)in PBS were added to each well and incubated for 4 h.After centrifugation,the precipitate of formazan blue was dissolved in 150 μl DMSO.The light absorbance of the DMSO was measured at 570 nm on a microplate reader.2 Study on the inhibitory effects of TIPP on breast cancer and CML cancer in vivoFive-week old female BALB/c-nu/nu mice(specific pathogen free)were injected subcutaneously with MCF-7 and K562 cancer cells.When the average volume of tumors in mouse was reached to 100 mm3,the nude mice with MCF-7 cell implantation were randomly divided into 6 groups,and the nude mice with K562 cell implantation were randomly divided into 3 groups,seven mice in each group.For breast cancer model,PBS,25 mg/kg TIPP,25 mg/kg TIPP combined with 0.1 mg/kg actinomycin-D,25 mg/kg TIPP combined with 7.5 mg/kg taxol,7.5 mg/kg taxol and 0.1 mg/kg actinomycin-D alone in 0.1 ml PBS were injected to 6 groups respectively.For CML model,the mice in group 1 were injected with PBS through tail vein,the mice in group 2 were injected with TIPP at a dose of 25 mg/kg in 0.1ml PBS,and the mice in group 3 were injected with actinomycin-D at a dosage of 0.1 mg/kg.The tumor volume was calculated every 3 days for 3 weeks.Results1 Anticancer activity of TIPP in vitroThe result of proliferation assay showed that TIPP apparently inhibited MCF-7 cell and K562 cell proliferation in a dose-dependent manner for 48 h.TIPP also significantly inhibited the survival rate of these two cancer cell lines.The results of anticancer effects of TIPP on the two different types of cancer cell lines proved that the TIPP had anticancer activity in vitro.2 Inhibitory effects of TIPP on breast cancer and CML cancerTIPP showed an apparent inhibitory activity on tumor growth of breast and CML cancer in vivo,which was consistent with the findings of cell proliferation assay in vitro.The inhibition rate of TIPP on MCF-7 was 54%,and the inhibition rate of K562 was up to 26%.In breast cancer model,the inhibition rate of TIPP combined with actinomycin-D was 58%,whereas 74%inhibition rate was noticed in TIPP combined with toxal group.3 Tumor apoptosis inducing activity of TIPPLiver and tumor tissue of breast cancer in xenograft models were subjected to H&E staining to examine the safety and apoptotic ability of TIPP.No obvious damage to liver tissue was found in both TIPP and toxal treatment groups,either alone or in combination,while a slight toxicity to liver was found in actinomycin-D treatment groups,either alone or in combination with TIPP.Remarkable apoptosis was found in the breast tumor tissue in the groups of TIPP alone and TIPP combined with toxal,while,mild necrosis was found in the tumor tissue in the groups of actinomycin-D alone and actinomycin-D combined with TIPP.Above results suggested that TIPP alone and in combination with taxol is a safe therapy for the treatment of cancer.Conclusions and significance(1)It was the first time to study the anticancer activity of TIPP in vitro.TIPP inhibited proliferation of breast cancer and CML cancer cells and induced significant growth inhibition in MCF-7 and K562 cells in a dose and time dependent manner.(2)It was the first time to study the inhibitory effects of TIPP on breast cancer and CML in vivo.TIPP effectively inhibited the tumor growth in CML and breast cancer mice models.(3)H&E staining results indicated that TIPP inhibited the breast cancer via inducing apoptosis of tumor cells.(4)H&E staining results of normal liver tissue indicated that TIPP is safe for liver.(5)The combination therapy of TIPP with other potent anticancer drugs for the treatment of breast cancer was performed for the first time.The results proved that the TIPP combined with taxol induced higher tumor inhibition(74%)than TIPP alone(54%)in breast cancer.