Role And Molecular Mechanism Of Aspirin In Sensitizing The Chemotheropeutic Effect Of Cisplatin In Colon Cancer

Objective:Colon cancer is the most common digestive system malignant tumor.Surgery and chemoradiotherapy are main therapeutic approaches to treat colon cancer.Cisplatin is one of the most potent chemotherapeutic agents for the treatment of a wide array of solid malignancies.Nevertheless,because of numerous reports in recent years about the notable toxicity and extensive resistance,the clinical use of cisplatin was severely restricted.Aspirin and some other non-steroidal anti-inflammatory drugs have been confirmed using as a chemopreventive and chemotherapeutic agent for a range of cancers including colon cancer.To explore whether aspirin can be used as adjuvant drug to synergize the inhibiting effect of cisplatin in the process of colon carcinogenesison,we design experimentations in vivo and vitro to verify the hypothesis that this combination therapy could prevent tumor growth effectively under taking a lower dose of cisplatin and relieve the toxic side effects of patients.Methods:1.MTT assay was used to detect the cell viability of human colon cancer cells SW620,Lo Vo,RKO and DLD-1 treated with different concentration of aspirin alone or the combination of different concentration of cisplatin and aspirin(2 m M)for 48 h.2.colony formation assay was taken to view the influence of the combination of aspirin and cisplatin on the the proliferation ability of human colon cancer cells.The changed morphology of human colon cancer cells treated with the combination of aspirin and cisplatin was observed.3.wound-healing assay and transwell assay were performed to detect influence of the combination of aspirin and cisplatin on the the migration and invasion ability of human colon cancer cells.The expression levels of main EMT related proteins weredetected by a western blot assay.4.FACS analysis and AO/EB staining assay were taken to determine the inducing of cell apoptosis in human colon cancer cells treated with the combination of aspirin and cisplatin.The expression levels of main apoptosis related proteins were detected by a western blot assay.Immunofluorescence assay of human colon cancer cells treated with the combination of aspirin and cisplatin for 48 h was taken to monitor cytochrome-c released from mitochondria.5.The expression levels of main PI3K/AKT?RAF-MEK-ERK signaling pathway related proteins were detected by a western blot assay.MTT assay was used to detect the cell viability of human colon cancer cell treated with the combination of aspirin and cisplatin for 48 h after a pre-treatment of PI3 K inhibitor LY294002 or MEK inhibitor U0126.6.The expression levels of main NF-?B signaling pathway related proteins in nucleus and cytoplasm were detected respectively by a western blot assay.Immunofluorescence assay was performed to monitor subcellular localization of p65 and p50.7.Colon cancer cells were injected subcutaneously to construct a nude mice tumor model to evaluate the influence of the combination therapy on the growth of xenograft tumors.The expression levels of main PI3K/AKT ? RAF-MEK-ERK and NF-?B signaling pathway related proteins in xenograft tumors were detected by western blot assay and IHC.Results:1.Aspirin synergizes the inhibiting effect of cisplatin on cell viability in colon cancer cells.With the combination of aspirin(2 m M)the IC50 value of cisplatin in colon cancer cells decreased obviously.2.Aspirin synergizes the inhibiting effect of cisplatin on cell proliferation in colon cancer cells.With the combination of aspirin and cisplatin,the morphology of colon cancer was changed significantly3.Aspirin synergizes the inhibiting effect of cisplatin on cell migration andinvasion in colon cancer cells.Main EMT related proteins were downregulated.4.Aspirin synergizes the inducing effect of cisplatin on cell apoptosis in colon cancer cells.The intrinsic mitochondrial apoptotic pathway was activated.5.Aspirin synergizes the inhibiting effect of cisplatin on PI3K/AKT ?RAF-MEK-ERK and NF-?B signaling pathway in colon cancer cells.6.Aspirin synergizes the inhibiting effect of cisplatin on tumor growth in vivo in a xenograft mouse model of human colon cancer by targeting PI3K/AKT ?RAF-MEK-ERK and NF-?B signaling pathway.Conclusion:This study showed that low-does aspirin could synergize the inhibiting effect of cisplatin on the initiation and development of colon caner in vivo and in vitro by targeting PI3K/AKT ? RAF-MEK-ERK and NF-?B signaling pathway.Aspirin as adjuvant drug combined with cisplatin could offer a new approach for the treatment of colon cancer.The combination therapy could relieve the toxic side effects of patients with the same therapeutic effect working.