Research On The Mechanism Of Compound GA Of Inhibitory Action On Lung Adenocarcinoma

Objectives:The morbidity and mortality of the patients who suffered lung cancer are very high and without remarkable treatment effect in recent years.GA is a new class of two terpene compounds with antitumor activity obtained from a series of gibberellin derivatives.We previously showed that compound GA have development inhibition to lung cancer cell(A549)growth.Based on our previous research,we will focus on lung cancer cell,A549,SPC-A-1 and XWLC-05,and take them as in vitro model,A549 and XWLC-05 as in vivo model,to evaluate the effect of GA and toxicity in nude mice.We take a look at some of the possibilities and potential clinical value of compound GA.This research is helpful to find the new drug for lung adenocarcinoma.Methods:Part ?:1.Choosing human lung adenocarcinoma cells,A549?SPC-A-1?XWLC-05,as research objects.Those three objects are treated with different concentrations of GA each other,then viability of cells are analyzed by to observe shape of cells and MTT assay.2.Anti tumor activities of compound GA are evaluated on mice bearing transplant sarcoma(S180)and on nude mice bearing transplant human lung cancer,A549 or XWLC-05,with the way of filling the stomach,based on inhibition rate/tumor growth inhibitory rate(%)and relative tumor growth rate(T/C%).Toxicity are preliminary assessed with spleen index or thymus index.Part ?:By retained samples of bone marrow,kidney and intestinal tissue of nude mice bearing transplant human lung cancer after in vivo experiment for viewing specifics toxicity of GA.Wright-Giemsa stain method are used in bone marrow samples to detect the hematopoietic function of nude mice.HE staining is used in kidney and intestinal tissue to observe organs injury.Part ?:1.Choosing human lung adenocarcinoma cells,A549?SPC-A-1?XWLC-05,as research objects.Character of apoptosis are assessed by Hochest33258 and TUNEL staining;The percentage of apoptosis are detected by flow cytometry analysis.Detection of mitochondrial pathway by western blot pattern:The symbolic protein expression in A549 cells and SPC-A-1 cells which after incubated with GA.Meanwhile,mitochondrial membrane potential changes are detected.Testing of endoplasmic reticulum pathway and death receptor pathway by western blot pattern:The symbolic protein expression of lung cancer cells.2.The pathological changes of transplant lung cancer(A549)influenced by compound GA are detected under electron microscope,especially apoptotic body.The expression of Caspase3?Caspase8?Caspase9 and TUNEL in the tumor tissues are measured by immunohistochemistry.Part ?:1.After lung cancer cells treated with different concentrations of GA and cis-platinum,viability of cells are analyzed by MTT assay;2.Antitumor activities of combined GA and cis-platinum are evaluated on mice bearing transplant sarcoma(S180),as well as on nude mice bearing transplant human lung cancer,A549 and XWLC-05,with the same way of administration and evaluation criterions.Results:1.In vitro and in vivo antitumor activity and toxicity evaluation of compound GA.1.1 As a result of treated by GA,human lung cancer cells,including A549?SPC-A-1?XWLC-05,are to be inhibited the proliferation and given rise to morphological changes.GA have stronger inhibitory effect on A549 and SPC-A-1 than XWLC-05.Compared with DDP,in heavy concentrations,GA have the same efficiency of tumor inhibiting.1.2 Compound GA obviously inhibited the tumor growth of S180 in mice.Both the dose of 3 mg/kg and 9 mg/kg by the way of filling the stomach indicated remarkable in vivo antitumor activity.It is also inhibited the tumor growth of nude mice xenograft of A549 and XWLC-05,and the T/C%are the 61.07%?59.77%?47.33%and 55.41%?41.97%?40.35%respectively at the dose of lmg/kg,3 mg/kg,9 mg/kg.1.3 Comparing with DDP,GA has lessen effect on immunity,body weight of mice and nude mice,without myelotoxicity,intestinal injury and kidney damage.However,DDP leads to kidney injury and weight loss.2.In vitro and vivo antitumor activity and toxicity evaluation of co-administration of compound GA and cisplatin(DDP).2.1.The results reflect co-administration of low dose of GA and DDP has distinct inhibited the proliferation of human lung cancer cells(A549?SPC-A-1?XWLC-05)by MTT assay,and it shows that two drugs act synergistic effect in some certain range of concentrations.2.2 Co-administration of low dose of GA and DDP has strongly inhibited the tumor growth of S180 in mice,as well as the tumor growth of nude mice xenograft of A549 and XWLC-05.It shows that two drugs paly additive action,particularly in the test of nude mice xenograft of XWLC-05.2.3 Co-administration of low dose GA and DDP is safety without any additional toxicity than that in the dose of 0.5mg/kg of DDP.3.The effects and mechanism of compound GA induces apoptosis of tumor cells in vivo and in vitro.3.1 The results indicated that GA can give birth to caspase 4,8,9 activities of lung cancer cell(A549,SPC-A-1),while the cleave-caspase9 be activated,Cytochrome C release,Bax and Bcl2 expression change by western-blot.2.GA contributes to activity of cleave-caspase4 and increase the expression lever of GRP78?GADD153 and ATF4,but cannot advance the activity of cleave-caspase8 of lung cancer cells.3.2 Apoptotic cells can be found in the xenograft lung cancer cells which treated by GA under electron microscope.It is stem from test,the expression of Caspase3,Caspase 8,Caspase 9 and TUNEL in xenograft lung cancer cells which treated by GA are higher than those without drug utilized.'Conclusions:1.Compound GA could inhibit the proliferation of human lung cancer cells,both in vivo and ex vivo.2.Compound GA obviously inhibited the tumor growth of S180 in mice,and nude mice xenograft of A549 and XWLC-05,with low toxicity,including without myelotoxicity,intestinal injury and kidney damage.3.Compound GA inducing apoptosis of lung cancer cells is related to the mitochondrial pathway and endoplasmic reticulum pathway,but may not be involved in the death receptor pathway.4.Co-administration of low dose of GA and DDP may act impactful role in clinical treatment with excellent action and low toxicity in the future,which offer more options to patients with DDP utilize limited.