The Effect Of Xien Yu Tang On Treating Sensitized Mice Against Lung Cancer Cells And The Study On Expression Of MMP-2 And TIMP-2

ObjectiveThe Chinese herbal decoction Xien Vu Tang(XYT)formulated by professor Chen, Rui-Shen on lung cancer was adopted to treat Lewis Lung Cancer(LLC)in order to investigate its ability to inhibit LLC cells invasion,mobility and metastasis.The lab researches look into:1.the effect of XYT to promote apoptosis,inhibit the proliferation,invasion and metastasis of Lewis lung cancer(LLC)cells,2.the expression of relevant genes for ECM degradation,biochemical activities between MMP-2 and TIMP-2 in Lewis lung cancer.3.the test animal model to evaluate the impacts by using Xien Vu Tang on caner cell's growth and mobility.Methods1.Groups:Control group(CG)and three Xien Yu Tang(XYT)test groups designated as XYTL=low dosage of XYT decoction group(1.092g/ml),XYTM=middle dosage of XYT decoction group(2.184g/ml),XYTH=high dosage of XYT decoction group (4.368g/ml).2.In Vitro:The Lewis lung cancer cells were thawed and cultured.During the culture of mouse Lewis lung cancer cells,the culture solution was replaced daily.Lewis lung cancer cells suspension was incubated in a 25ml bottle with density as 1.0×10⁵/ml.Cells were divided into 4 groups(CG,XYTL,XYTM & XYTH).After a 48-hour treatment,the cells of each group were collected and the flowing measures were carried out.Cellular proliferation and growth activities were assayed by MTT colorimetry;Cell cycle distribution and apoptosis rate were determined by Flow Cytometer(FCM);Boyden Chamber was used to analyze Xien Yu Tang against the adhesion,invasion and migration of Lewis lung cancer cells.3.In Vivo:The Lewis lung cancer cells were thawed,inoculated and injected to BALB/C mice.The subcutaneous injection was given on the inner side of the left rear leg.The mice were grouped randomly in 4 groups(CG,XYTL,XYTM & XYTH), 10 mice per group.On 2^ndday after tumor cells suspension injecting,the mice were fed with 3 different decoctions of XYT on 3 test groups daily for 14 days.Daily observe the appearance in all 4 mice groups.After 24 hours on last feeding,all mice were sacrificed by cervical vertebra dislocation.The tumors were removed and weighed to get the average weight and determine the inhibition rate of tumors growth.Check the histopathologic change of tumor tissue with HE staining technique.Use SABC immunohistochemical analysis to measure the activity of MMP-2 and TIMP-2.4.All lab data were analyzed with SPSS computer software.The results expressed as mean±standard deviation((?)±s),Use Anova method to compare the data between groups.Results & DiscussionThe following results were observed and recorded by comparing the control group(CG)with the 3 XYT test groups(XYTL,XYTM,XYTH):1.By appearance:At the end of test period,the mice without being treated by XYT were smaller in size and less active.They were observed to have lost more hair,become less lustered and to be more gregarious than other groups.2.The observation on suppression in tumor cells growth:Tumor suppression rate =(1- average tumors' weights of test groups/average tumors' weights of control group)×100%.Tumor suppression rates were as at XYTL group=32.31%,at XYTM group=36.25%, at XYTH group = 40.88%.There was an inverse proportion between tumor weight and herbal dosage(P＜0.01).The higher dosage added,the smaller of tumor in weight with a higher inhibition rate of tumor growth.3.The morphological observation and density on the growth of mouse Lewis cancer cells:The tumor cells grew condensed with clear appearance.While the groups treated with XYT,the tumor had low density,the spaces between cells were bigger,cells were smaller in size,some cells became corrugated and flew up,and some cells became less pervious to light.The lab test proved that XYT can suppress the growth of tumor,promote apoptosis.By increasing the XYT dosage,more cells became corrugated and flew up.4.Hematoxylin and Eosin Stain finding:The tumor on the group without being treated by XYT had typical cancer cell shape with inconsistency in size and appearance,the cells had bigger nucleus with increased rate of nucleus/plasma;the size,shape and dyed density were inconsistent between nuclei,the nucleolus was enlarged.The number of lymphocyte was reduced in intercellular substance.On the test groups treated by XYT,the cell apoptosis was found in tumor tissue,the lymphocytes invasion could be seen in the intercellular substance.5.Measuring MTT-OD value of all groups:The groups have been treated by XYT that had lower MTT-OD value.The suppression rate of tumor cell proliferation =(1 - light absorption value of test group / light absorption value of control group)×100%.The higher XYT dosage treated,the higher suppression rate has been presented which XYTL =12.12%,XYTM = 16.39%and XYTH = 21.15%respectively.6.The assay of cancer cell mobility with modified Boyden Chamber technique, we found that all XYT groups can suppress LLC invasion.The mobility/invasion for the groups were XYTL=130±15%,XYTM=97±11%and XYTH= 85±9%.The XYTM and XYTH groups showed significant suppression(P＜0.01).7.The finding of cell cycle and apoptosis of cancer cells with FCM assay: Compare the test groups with control group,the cell rate was up-regulated on G₀/G₁ phase in all 3 XYT test groups(P＜0.01).On S phase,the cell rate has been much reduced in the three test groups and there was dosage-depended and had obvious diversity among the test groups.The mean difference is significant(P＜0.01).On G₂/M phase,no significant difference between groups (P＞0.05).Those findings evince XYT can interrupt,delay or stop the cell cycle in turn to inhibit the tumor growth,the higher dosage of XYT,the better effect.Also we found that XYTM and XYTH may apparently promote the apoptosis of LLC cells,this is to confirm the lab finding on the morphological observation and density on the growth of mouse Lewis lung cancer cells.8.By applying SABC Immunohistochemical Analysis,the density of MMP-2 and TIMP-2 can be determined in control group and 3 test groups.Comparing with Lewis lung carcinoma:the decreased expression of MMP-2 and increased expression of TIMP-2 were significant(P＜0.01)in all 3 XYT groups. Comparing CG and XYTH,the elevation in TIMP-2 were obviously significant (P＜0.01)in XYTH.Meanwhile a negative relation was noted in TIMP-2 and MMP-2 expressions during carcinogenesis.There was a negative relationship between TIMP-2 expression and Lewis lung cancer metastasis,which showed the close relationship between MMP-2 and metastasis in advanced mouse Lewis lung carcinoma(P＜0.01).ConclusionMetastasis is the major cause of death at cancer patients.Many various treatment strategies have focused on preventing the occurrence of metastasis, which include the cell adhesion,degradation of the extra cellular matrix(ECM) and changing of the cell motility,etc.Therefore,the development of new remedies or the utilization of natural medicines targeting at metastasis has been interested and studied extensively.At present time many Chinese herbs have been proved to have the power of anti-inflammation,anti-allergic and anti-cancer activeness.Some of the active components from herbs had been isolated and identified.Recently,a lot of reports regarding Chinese herbs in studies of variety of anti-carcinogenesis,apoptosis and suppressing cell proliferation have been published.The lab results are written below:1.The anticancer mechanisms of the active ingredients are to effectively Promote apoptosis,inhibit the proliferation,invasion and metastasis of cancer cells.XYT decoction can inhibit the growth of Lewis lung carcinoma in mice & the cell proliferation,effectively promote apoptosis,suppress invasion and metastasis of Lewis lung cancer cells.The effect might be associated with inhibition of cell cycle and the initiation of apoptosis on cancer cells.2.The relation between MMP and tumor cell growth,invasion,and metastasis is directly proportional.Proteolytic activity of MMP is inhibited by specific inhibitors,the tissue inhibitors of matrix metalloproteinase(TIMP).Under the cancer development,the expression of MMP and TIMP is highly coordinated. The excess degradation and disruption of basement membranes by activated MMP-2 may be a key step in inducing lung cancer cells' invasion and metastasis.The imbalance between MMP-2 and TIMP-2 may be also a critical factor that affects biologic behavior of lung carcinogenesis,invasion and metastasis.XYT decoction can suppress the Lewis lung cancer's invasion and metastasis that might be associated with the decrease of MMP-2 level and the increase of TIMP-2 level.3.In nontumor tissues that presumably express low levels of MMP-2 and MT1-MMP, even low amount of TIMP-2 is sufficient to inhibit MMPs activation.The activity of MMP-2 activation initially increases when TIMP-2 increases until TIMP-2 levels reach the optimum for maximal MMP-2 activation.However,very high levels of TIMP-2 expression has the inhibitory capability to both MMPs activity and MMP-2 activation,that is due to the excessive TIMP-2 binding to MT1-MMP as well as direct binding to MMP-2.XYTH decoction could significantly elevate the TIMP-2 level in Lewis lung carcinoma,therefore the lung-cancer cells have been inhibited to reproduce in turn that the XYT could inhibit the tumor cellular responses including growth,proliferation,differentiation,migration,metabolism and survival. The lab tests also indicated that the XYT could reduce the invasion and metastasis of lung cancer cells in vivo and in vitro,thereby constituting an adjuvant treatment for metastasis.On this study we provided a putative mechanism of how the lung cancer cell proliferation,adhesion,invasion and metastasis,and we might be benefit to develop a modified XYT formula for a target therapy and new concept of modulating herbal medicine in the future.