Inhibitory Effect On IL-6/STAT3Pathway, A Possible Mechanism By Which Pien Tze Huang Treats Colorectal Cancer

Colorectal cancer (CRC) was one of the most common human malignant gastrointestinalcancers with over1.2million new cases and608,700deaths according to the statistics in2011. In China, the morbidity and mortality of CRC are increasing in recent years.Recently, the diagnosis, surgery, radiotherapy and chemotherapy in CRC have madesome progress. However, the problem of metastasis and recurrence has not been solved yet.Pien Tze Huang (PZH) is a well-known traditional Chinese formulation first prescribed bya royal physician450years ago during the Ming Dynasty. The main ingredients of PZHinclude Moschus, Calculus Bovis, Snake Gall, and Radix Notoginseng, all of which havefunctions of heat-clearing, detoxifying, blood circulation-promoting, blood stasis-andswelling-reducing, and pain-relieving. PZH has exhibited therapeutic effects in clinicaltrials of tumors, especially gastrointestinal carcinoma. However, the mechanism of itsanticancer activity still remains largely unknown. Therefore, in this study, we evaluated themolecular, cellular and animal effects of PZH which could provide a reasonable, accuratetheoretical basis for clinical.CRC has been proved to be close related with cellular proliferation and apoptosis. Signaltransducer and activator of transcription3(STAT3) is an important transcription factor thatplays an essential role in cell survival and proliferation. STAT3can bind to IL-6called asIL-6/STAT3. It is known that over-expression of Cyclin D1, Bcl-2and others, mediated bythe abnormal activation of IL-6/STAT3, will lead to excessive cell proliferation andapoptosis resistance, which maybe cause tumorigenesis.As a regulatory factor, Cyclin D1is a nuclear protein associated with cell cycle, and isnecessary in cancer development. The cell proliferation is primarily regulated by the G1/Stransition, one of the main checkpoints in cell cycle, which is highly mediated bypro-proliferative Cyclin D1and Cyclin-dependent kinase4(CDK4). However, Cyclin D1expression is regulated by the expression and activation of STAT3. In normal cells, CyclinD1is strictly controlled. But over-expression of Cyclin D1can shorten G1period, reducethe dependence of cells on the mitogen, and promote the proliferation of cells. Bcl-2familyproteins include anti-apoptotic members such as Bcl-2and pro-apoptotic members such asBax. Bcl-2is an important factor in the regulation of apoptosis by blocking the apoptoticprocess with a multi-stage, multi-level manner. Higher Bcl-2-to-Bax ratio by aberrantexpression of the proteins is found commonly in various cancers. Proliferating cell nuclear antigen (PCNA), an acidic nuclear protein, has been recognized as a histologic marker forthe G1/S phase in the cell cycle and is closely associated with cell proliferation and DNAreplication. And p21is a proliferation inhibitor, which can alter the function of PCNA tosuppress cell proliferation. Caspases-3, represented by a family of cysteine proteases, is akey executioner of apoptosis. Caspase-3is known to be essential for apoptosis-associatedchromatin margination, DNA fragmentation, and nuclear collapse.Angiogenesis, a physiological process involving the growth of new blood vessels fromthe pre-exising vasculature, is crucial to wound healing, reproduction and embryonicdevelopment. However, deregulated angiogenesis plays a crucial role in the developmentof cancer. When the tumor grows to a certain size, oxygen delivery by diffusion is nolonger sufficient, causing tumor cells to induce the sprouting of new blood vessels frompre-existing vasculature, creating a blood supply system within the tumor that is essentialfor continued tumor growth as well as providing a pathway for hematogenous metastasis.Vascular endothelial growth factor (VEGF) is considered as one of the strongest stimulatorof angiogenesis. VEGF is highly expressed in a wide variety of human tumor, which hasbeen associated with tumor progression, invasion and metastasis, and is poorer survivaland prognosis in patients. STAT3pathway is also concerned with the development ofangiogenesis in tumor. Activation of STAT3pathway would induce the angiogenesis oftumor. So, in this study, MTT assay, flow cytometry, RT-PCR, Western blotting, ELISA,Bio-plexes and other detection methods were used to detect the effect of PZH on theproliferation, apoptosis, angiogenesis both in tumor cell and in animal model, and theinfluence on IL-6/STAT3pathway. The result would provide a theoretical basis for thePZH in CRC treatment.Part Ⅰ: The study of PZH on the proliferation and apoptosis in HT-29cells and IL-6/STAT3pathwayObjective: To explore the effect of PZH on the proliferation and apoptosis in HT-29cells and IL-6/STAT3pathway. Methods: The HT-29cells were treated with differentconcentration of PZH (250μg/ml,500μg/ml and1000μg/ml) for different periods of time(1,3,6,12and24h). MTT assay, flow cytometry, RT-PCR, Western blotting, and othermethods were used to detect cell proliferation, apoptosis and the level of STAT3phosphorylation. Results: PZH inhibited the proliferation and induced the apoptosis ofcells, and changed the level of IL-6/STAT3signaling pathway in HT-29cells. Conclusion:The effect of PZH on anti-proliferation and apoptosis induction was related to down-regulate the expression of Cyclin D1, CDK4, PCNA, p21, Bcl-2, Bax, Caspase-3and affected activation and phosphorylation of STAT3.Part Ⅱ: The mechanism study of PZH on anti-angiogenesisObjective: To explore the mechanism of PZH on anti-angiogenesis. Methods: Chickenchorioallantoic membrane (CAM), human umbilical vein endothelial cells (HUVECs) andHT-29cells were selected as experimental subjects. MTT assay, FCM, RT-PCR, ELISA,and other methods were used to detect the indexes of angiogenesis, cytokines level, and soon. Results: PZH inhibited angiogenesis of CAM, inhibited proliferation, migration, andtube formation of HUVECs, and reduced VEGF expression of HT-29cells and HUVECs.All of these were dose-dependent. Conclusion: The anti-vascular endothelial cellproliferation and inhibition of VEGF expression effects of PZH may be related to STAT3signaling pathway.Part Ⅲ: The mechanism of PZH on the growth inhibition of nude micewith HT-29xenograft tumorObjective: To explore the effects of PZH on cell proliferation, apoptosis, angiogenesisand IL-6/STAT3pathway in animal model. Methods: Animal model of transplanted tumorwas established. The mice were given intra-gastric administration of0.234g/kg/d dose ofPZH in treatment group and10ml/kg/d dose of saline in control group. In order to verifythe effect of PZH on nude mice with HT-29xenograft tumor, RT-PCR,immunohistochemistry, Western blotting, Bio-plexes suspension array and other methodswere used and cell proliferation, apoptosis and angiogenesis were deceted. Results: PZHinhibited the tumor growth, cell proliferation and angiogenesis, and induced cell apoptosis.The impact on IL-6/STAT3signal pathway was changed after the intervention of PZH.Conclusion: The molecular mechanisms of PZH may be related to IL-6/STAT3pathway.