Mechanism Of Andrographolide Derivative ADA Inhibiting Metastasis Of Human Breast Cancer Cells

Breast cancer is one of the malignant tumors that seriously threaten women's health.According to statistics,the number of breast cancer deaths in the world accounts for 11.6% of the total cancer deaths,and the mortality rate remains high,which is the second leading cause of cancer deaths.Tumor metastasis is the main cause of death of breast cancer patients.Studies have shown that once breast cancer cells leave the primary site,it will be difficult to cure.Chemotherapy is a commonly used treatment method in clinical practice.Although it can promote tumor regression,there is still the possibility of recurrence and metastasis.At the same time,it will be accompanied by many side effects,such as bone marrow suppression,gastrointestinal reactions and heart damage.At present,many natural medicines have great potential in anti-cancer,and have the advantages of rich variety and small adverse reactions.Therefore,searching for natural medicines for the treatment of breast cancer metastasis is an important direction in the research and development of new drugs.Andrographis paniculata is derived from the dry aerial part of Andrographispaniculata?Burm.f.?Nees,a herbaceous plant in the family Cymbidium.It has the effects of clearing away heat and detoxifying,cooling blood and reducing swelling.Modern pharmacological studies have shown that andrographis paniculata has anti-tumor,anti-inflammatory,hepatoprotective and anti-infective effects.Its active ingredient is a diterpene lactone compound represented by andrographolide.Andrographis paniculata can not only inhibit tumor metastasis,but also exert anti-tumor effects through various ways.It can also be combined with other anti-tumor drugs to reverse multidrug resistance.It is a potential new drug candidate.The research group synthesized a series of andrographolide derivatives in the early stage,of which the andrographolide derivative ADA has the strongest antitumor effect.The study found that andrographolidederivative ADA can cause the activation of caspase family?increased expression of caspase3 and cytochrome C?in MCF-7 cells,and can also regulate the expression of Bcl-2 and Bax proteins.In vivo pharmacodynamic experiments show that ADA can significantly reduce the size and weight of tumors,and the tumor inhibition rate is more than 80%.In addition,the research group also found that andrographolide has a significant inhibitory effect on TGF-?₁ secreted by murine breast cancer 4T-1 cells.In this paper,human breast cancer MDA-MB-231 and MCF-7 cells are used as models to study the mechanism of ADA inhibiting breast cancer cell metastasis.MTT method was used to detect the inhibitory effect of ADA on the proliferation of two types of cells.The scratch experiment,Transwell experiment and Invasion experiment examined the effect of ADA on the migration and invasion of two cells after 24 h.Western Blot experiment was used to detect the expression of E-cadherin,N-cadherin,MMP-2 and MMP-9 protein in MDA-MB-231 and MCF-7 cells,as well as TGF-?₁,Smad2/3 and Smad4 protein expression in the TGF-?₁/Smad signaling pathway.Fluorescence quantitative PCR experiment was used to detect the effect of ADA on MDA-MB-231 and MCF-7 cells after 48 h,on the expression levels of E-cadherin m RNA and MMP-9 m RNA in the two cells.MTT experimental results show that After ADA treatment of human breast cancer MDA-MB-231 cells and MCF-7 cells for 24 h,the proliferation of both tumor cells was significantly inhibited,and has a significant dose-dependent,and their IC50 values are 9.63 ± 1.96 ?g/ m L and5.12±0.14 ?g/m L?P<0.01?.The inhibitory effect of ADA on the proliferation of two types of cells is significantly better than that of its lead compound A,indicating that ADA has a stronger inhibitory effect on breast cancer cell proliferation than A.The results of the scratch experiment showed that compared with the blank control group,after24 h of MDA-MB-231 cells treatment with ADA at 8 and 10 ?g/m L,the cell migration rates respectivelywere 12.84% and 8.92%?P<0.01?;After 24 h of MCF-7 cells treated with ADA at 6,8 and 10 ?g/m L,the cell migration rates respectively were 12.36%,10.37%,and 9.15%?P<0.01?.Transwell experimental results showed that compared with the control group,the migration inhibition rates of 8 and 10 ?g/m L ADA on MDA-MB-231 cells were 62.5% and 68.4%,respectively?P<0.01?;The migration inhibition rates of MC-7 cells by of 6,8,and 10 ?g/ m L ADA were 29.9%,43.1% and 57.7%?P<0.01?.Invasion experiment results show that: each ADA dosing group can inhibit the invasion of MDA-MB-231 cells,and their invasion inhibition rates are 25%,42.7%,59.3%,72.8% and 79%?P<0.01?;.After treatment with 6,8and 10 ?g/m L ADA for MCF-7 cells for 24 h,compared with the control group,their invasion inhibition rates were 66.4%,74.3%,and 78.6%,respectively?P<0.01?.It shows that ADA can inhibit the migration and invasion of MDA-MB-231 cells and MCF-7 cells.Compared with A,ADA has a stronger effect.The results of Western Blot experiments showed that after 8 and 10 ?g/m L ADA were treated on MDA-MB-231 cells for 48 h,the expression of E-cadherin in the cells was significantly increased?P<0.01?;while different concentrations of ADA treated MCF-7 cells After 48 hours,the expression of E-cadherin increased,but there was no significant difference.After treating MDA-MB-231 cells with 6,8and 10 ?g/m L ADA for 48 h,the expressions of N-cadherin,MMP-2,MMP-9,TGF-?₁ and Smad4 proteins all decreased significantly?P<0.01?;10 ?g/m L ADA can reduce the expression of N-cadherin,MMP-2,MMP-9,TGF-?₁ and Smad4 protein in MCF-7 cells after 48 hours?P<0.05,P<0.01?.In addition,48 hours after ADA treated MDA-MB-231 cells and MCF-7 cells,the levels of Smad2/3 protein in the two cells did not change significantly.It is speculated that ADA may be involved in inhibiting the phosphorylation level of Smad2/3 protein,thereby inhibiting Smad4 protein.expression.The results of fluorescent quantitative PCR experiments showed that compared with the blank control group,8 and 10 ?g/m L ADA treatment of MDA-MB-231 cells for 48 h can significantly increaseE-cadherin m RNA,reduce MMP-9 m RNA expression,and its amplification The multiples were 3.3 times,6.9 times,and 0.42 times,0.2 times?P<0.05,P<0.01?.After 48 h of ADA acting on MCF-7 cells,10?g/m L ADA can increase the expression of E-cadherin m RNA,and its amplification factor is 3.7 times?P<0.05?;8 and 10 ?g/m L ADA can inhibit MMP-9 The expression of m RNA has an amplification factor of 0.8 and 0.7 times?P<0.01?.In summary,andrographolide derivative ADA can inhibit the proliferation and metastasis of MDA-MB-231 and MCF-7 cells.The mechanism may be the regulation of E-cadherin and MMP-9 in MDA-MB-231 cells and MCF-7 cells.The expression of m RNA and protein levels can suppress the expression of MMP-2 and N-cadherin,and then regulate the process of EMT,thereby inhibiting cell migration.At the same time,ADA participates in inhibiting the metastasis of breast cancer cells by inhibiting the expression of TGF-?₁ protein and Smad4 protein in the TGF-?₁/Smad signaling pathway.