Lung Cancer-suppressing Mechanism Of Wound-healing Microenviroment And Drug Therapy

The occurrence and development of tumor is closely related to the tumor microenvironment, and tumor is called the wound that never healed. There are many similarities between the progress of wound-healing, which is thought to be one of the causes of cancer, and the occurrence and development of tumor in signal transduction. However,wound-healing is the basic requirement for functional recovery of tissue and organ, it has not been reported that whether wound-healing can prevent tumor progression. From the view of traditional Chinese medicine that “ wound healing requires sufficient vigor ”,we propose a scientific hypothesis that wound-healing microenvironment of could prevent the occurrence of cancer. To prove whether this hypothesis is correct or not, we study lung cancer-suppressing mechanism of wound-healing microenviroment and drug therapy.First, we screened out wound-curing drugs by the full-thickness skin excision model in mice, the characters of wound-curing drugs were observed by TPA-induced ear edema, macrophage phagocytosis and blood perfusion volume, and wound-healing drugs are combined based on the characters of wound-curing. Then, in vitro experiment,we use combinatory wound-healing drug to treat A549 cells?BEAS-2B cells?ECV304 cells and L929 cells. MTT assay was used to examine cell proliferation, scrape loading and dye transfer were used to observe the gap functional intercellular communication(GJIC), the number of tumor spheres and soft agar colonies were used to test the cell self-renewal ability. At last, we observed effects of combinatory wound-healing drug-builted wound-healing microenvironment on lung cancer progression by urethane-induced lung cancer model in mice. The impacts of combinatory wound-healing drug on the normal physiology were examined by body weight and autonomic activity, and lung pathological changes, lung cancer incidence and pulmonary tumor nodules were also observed., Immunohistochemical analysis was used to test ki67(cell proliferation marker) and cleaved caspase-3(apoptosis related marker), western blot was used to test Oct4 and Nanog(tumor stem cell marker), Connexin43(intercellular communication marker), E-Cadherin, N-Cadherin, and vimentin(epithelial mesenchymal transformation markers). In addition, to further explore cancer-suppressing mechanism of wound-healing microenviroment, we also observed effects of pulmonary inflammation, macrophages depletion and lung injury induced by lipopolysaccharide tracheal instillation, liposome-encapsulated clodronate intraperitoneal injection and bleomycin tracheal instillation respectively on lung cancer progression and combinatory efficacy of wound-healing drugs by urethane-induced lung cancer model in mice, observation was focused on TNF? and MPO(cell inflammatory markers), ROS and 8-OHdG(cell damage markers) by ELISA kits.The results showed that shikonin,notoginsenoside R1 and aconitine among the screened drugs could promote wound recovery in the full-thickness skin excision wound in mice. In laser speckle imaging, significantly greater blood flow was detected in the wound areas in the notoginsenoside R1 and aconitine groups relative to the control group. Only shikonin suppressed TPA-induced ear edema effectively. The results of the macrophage phagocytosis assay showed that notoginsenoside R1 and aconitine considerably enhanced the macrophage phagocytosis. Therefore, the three drugs were combined into wound-healing compound to form a wound-healing microenvironment and to observe the influence of wound-curing microenvironment on tumor development in vivo and vitro.In vitro experiment, 0.2?M~1?M shikonin suppressed ECV304 cell proliferation, 2?M~10?M notoginsenoside R1 suppressed L929 cell proliferation and facilitated ECV304 cell proliferation, and 0.02?M~0.1?M aconitine facilitated L929 cell proliferation. Combinatory wound-healing drug properly promoted ECV304 cell and L929 cell proliferation but didn't suppress A549 cell proliferation, which led to restoration of the urethane-decreased intercellular communication in BEAS-2B cells and suppression of the soft agar colonies in A549 tumor cells.In the urethane-induced lung cancer model in mice, the inhibitory rate of lung cancer incidence was 50%, 30% and 35%, in 0.2 mg/kg aconitine, 2 mg/kg shikonin, 20 mg/kg of notoginsenoside R1 group respectively, whereas it was as high as 90% in combinatory wound-healing drug group. Pathological results showed that the three drugs used alone could only certainly improve lung tissue pathology, whereas combinatory wound-healing drug could nearly restore lung tissue pathology to normal physiology. Immunohistochemical results showed that combinatory wound-healing drug could down-regulate the expression of ki67 and up-regulate the expression of Cleaved Caspase-3. Western showed that combinatory wound-healing drug could down-regulate the expression of Oct4 and Nanog(stem cell markers), up-regulate the expression of Connexin43 and E-Cadherin, and reduce N-Cadherin and vimentin. In addition, we find that lipopolysaccharide tracheal instillation mainly increased MPO and TNF? in lung but didn't obviously promote lung cancer progression in mice, bleomycin tracheal instillation mainly increased ROS and 8-OHdG in lung but promoted lung cancer progression, and that liposome-encapsulated clodronate depleted the macrophages before lung carcinogenesis to show a anti-inflammatory effect indicated by decreased TNF? and MPO and a lung injury-promoting efficacy indicated by increased ROS and 8-OHdG, which promoted lung cancer progression. Notably, lung cancer preventive efficacy of combinatory wound-healing drug-builted wound-healing microenvironment could be decreased under above three state.Above all, our findings suggest that wound-healing microenvironment can prevent lung cancer development and progression and combinatory wound-healing drug might be a promising measure for lung cancer prevention and therapy.