| Malignant tumor, a devastating disease, is a great threaten to the health andlife of human. Although several novel strategies or drugs have been emerged,cytotoxic chemotherapy is the mainstay of medical approaches to treat cancer.Cisplatin, which inhibits cell proliferation and induces cell cycle arrest byforming interstrand and intrastrand DNA cross-links, is one of the most widelyused chemotherapeutic agents in the treatment of cancer. However, the efficacyof cisplatin based on the treatment is limited in curing most tumors due todose-dependent toxicity and development of cisplatin resistance. Therefore, itwould be highly desirable to develop new approaches that would overcome thedrug resistance and lower the doses administered to the patient.Angiogenesis, the process leading to the formation of new blood vessels,plays a central role in the survival of cancer cells, in local tumor growth, and inthe development of distant metastasis. As a strategy for cancer therapy,antiangiogenic therapy attempts to stop new vessels from forming around atumor and break up the existing network of abnormal capillaries that feeds thecancerous mass. Some angiogenesis inhibitors are being conducted in a series ofclinical trials and unfold an inspiring perspective. Honokiol, as a potentantiangiogenic compound, has been used in antineoplasm in several tumor cell lines and in tumor model in mice. Antiangiogenic therapy, although effective ininhibiting tumor growth, has not been shown tumoricidal in most studies.However, this therapeutic limitation can be overcome by combiningangiogenesis inhibitors with cytotoxic therapies.Apoptosis (also named as programmed cell death), is strictly regulated bysome genes. In molecular oncology community, studies have shown thatapoptosis play an important role in the generation, development and regressionof tumor. Recently, apoptosis has been a target of anticancer drugs, and one ofthe ideal cancer therapy methods is to selectively induce tumor cells to apoptosis,sparing from normal cells at the same time. PNAS-4, as a protein activatedduring the early response to DNA damage, can induce significant apoptosis whenoverexpressed in osteosarcoma U2OS cells and unfold as a inspiring target geneof cancer gene therapy.In the current study, on the basis of mPNAS-4 gene sequence and itsderivative protein sequence, we first cloned the encoding region of mPNAS-4,then we constructed the eukaryotic expression plasmid—pcDNA3.1-mPS. Wefound that overexpression of mPNAS-4 had in vitro and in vivo antitumor effectsvia induction of apoptosis. Based on these results, two hypothesis in this workwere offered as follows: (1) since PNAS-4 can cause a significant increase in celldeath via apoptosis when overexpressed in some tumor cells, indicating thatPNAS-4 may be involved in the apoptotic response to DNA damage, and theknown result that the cytotoxic action of cisplatin against tumor cells is mediatedby DNA damage, the synergistic or enhanced antitumor effects may by obtainedwhen mPNAS-4 gene therapy was combined with low-dose cisplatinchemotherapy; (2) since overexpression of PNAS-4 can cause a significantincrease in cell death via apoptosis and the antiangiogenic and antitumor activityof honokiol, the synergistic or enhanced antitumor efficacy may by obtained inthe combination therapy with honokiol as an antiangiogenic compound andmPNAS-4 as a cytotoxic agent for carcinoma. To address the two hypothesis, we decided to investigate the antitumoreffects in vitro and in vivo when mPNAS-4 was combined with low-dosecisplatin or honokiol, respectively. In vitro, the additive proliferation inhibition ofLL2 and/or CT26 were obtained through the enhanced induction of apoptosiswhen mPNAS-4 was combined with low-dose cisplatin or with low-dosehonokiol, as evidenced by MTT assay, flow cytometry analysis, DNA ladder andco-transfection analysis, respectively. In the following animal expriments, weinvestigated the therapeutic efficacy of the combination treatment with mPNAS-4and low-dose cisplatin (or honokiol) against the LL2 lewis lung cancer and CT26colon carcinoma. Our data has shown that the single treatment with mPNAS-4gene therapy, cisplatin or honokiol has shown an antitumor efficacy, however, thecombination therapy has shown an enhanced antitumor efficacy. A moresignificant reduction in mouse xenograft tumor volumes and a more remarkableincrease of life span in the combination groups are observed, as compared withthe single treatment.To elucidate the precise mechanism by which mPNAS-4 plus cisplatincauses an additive antitumor effects, apoptosis and necrosis in the tumor tissueswere detected by TUNEL and H&E staining, respectively. The results showedthat the enhanced induction of apoptosis might be the principal reason for theimprovement of antitumor effectiveness in vivo. To elucidate the precisemechanism by which mPNAS-4 plus honokiol causes an enhanced antitumorefficacy, the alginate-encapsulated tumor cells assay and CD31immunohistochemistry staining were used to evaluate angiogenesis. Whileapoptosis in the tumor tissues were detected by TUNEL. The results also showedthat the improved inhibition of angiogenesis and the enhanced induction ofapoptosis might be the two principal reasons for the improvement of antitumoreffectiveness in vivo.Taken together, our data suggested that the combination therapy withmPNAS-4 gene therapy and low dose cisplatin (or honokiol) chemotherapy had an improved therapeutic efficacy. Therefore, our results may provide novel andeffective ways in the treatment of cancer. |
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