Effect Of Andrographis Effective Parts On MCF-7/ADM And Its Mechanisms

In our country, breast cancer is the most malignant tumor affecting adult women. Chemotherapy is one of the common methods for its treatment. However, the development of multidrug resistance(MDR) has been a huge challenge for clinicians to manage breast cancer therapy. Several mechanisms contribute to MDR including reprogramming of cellular metabolism, and it has become clear that the altered metabolism confers a growth and survival advantage to cancer cells. Unfortunately, the dysregulated metabolism in therapeutic resistance, which is a highly clinical relevant area in cancer metabolism research, has not been specifically addressed.In addition to chemical medicines, traditional Chinese medicine, which is known for its low toxic and effective quality, is commonly used for the treatment of cancers in China. Andrographis paniculata(Burm.f.) Nees(AP) is an important medical plant and its aerial part is commonly used in Chinese medicine. Diterpene lactones abundant in the aerial part are the material foundation for a broad range of cancers. Andrographis effective parts(AEP) was enriched by supramolecular technology in our previous work. The purpose of this paper was to investigate the effect of AEP on breast cancer cell line MCF-7/ADM cells and its mechanisms.The anti-proliferation effects on MCF-7 and MCF-7/ADM cells of AEP, andrographolide(A), 14-deoxy-11, 12-didehydroandrographolide(DDA), 14-deoxyandrographolide(DA), neoandrographolide(NA) and the mixture of above constituents(AMP) were assessed by MTT assay. Cell morphological changes of MCF-7 and MCF-7/ADM cells were observed by light microscope and high content screening(HCS) as well as fluorescence microscope after DAPI staining and AO/EB double staining. Cytometric analysis with Annexin V-FITC/PI double staining was applied to qualify the apoptosis and necrosis induced by AEP. Western blot analysis was done to investigate effects of AEP on the expression of PKM2, FASN, procaspase-3 and the ratio of Bcl-2/Bax.MTT assay demonstrated that A and AEP exerted potent anti-tumor effect on MCF-7 and MCF-7/ADM cells in a concentration-dependent and time-dependent manner. The IC50 values of A were 7.63±1.24, 4.96±1.00 ?g/mL(48 h) and 3.81±0.79, 2.52±0.18 ?g/mL(72 h) respectively. The IC50 values of AEP are 28.24±1.68?14.55±2.15 ?g/mL(48 h) and 17.08±1.68, 8.10±1.23 ?g/mL(72 h). Compared with AEP, anti-tumor effects of DDA, DA, NA and AMP on MCF-7 cells had no statistical significance(P>0.05). When it came to MCF-7/ADM cells, however, the anti-tumor effect of AEP was more powerful than that of DDA, DA, NA.Morphological changes of MCF-7 and MCF-7/ADM cells were evaluated successively. Cells grown in medium alone appeared spindle-shaped with dense intracellular gaps and an active proliferative capacity and cell nuclei were stained with a less bright fluorescence. After exposed to AEP for 48 h, cells exhibited a ring-shaped appearance, had a lower viability and fewer adhered cells. At the same time, marked morphological changes of cell nuclei such as condensation of chromatin, fragmentation to multiple aggregate of apoptotic bodies and nuclei stained with a bright fluorescence were found clearly. When the concentration of AEP ranging from 15 ?g/mL to 40 ?g/mL AO/EB double staining results showed necrotic orange chromatin was diffusely distributed, which indicated the necrosis of MCF-7/ADM.Results of Annexin V-FITC/PI double staining experiment were in accordance with that of DAPI staining experiment and AO/EB double staining experiment. Moreover, significant necrosis of MCF-7/ADM cells were detected when treated with 40 ?g/mL AEP for 48 h and the percent of necrotic MCF-7/ADM cells was 42.80%.Western blotting results showed that AEP at low concentrations(5, 10, 15 ?g/mL) could not downregulate the expression of PKM2, FASN and procaspase-3. When exposed to higher concentrations such as 20 ?g/mL and 40 ?g/mL, the expression of PKM2, FASN and procaspase-3 decreased significantly. Amazingly, it was at the concentration of 5 ?g/mL that AEP could notably reduce the expression of those three enzymes. However, AEP had a slight influence on the ratio of Bcl-2/Bax decreased dramatically.In conclusion, AEP could inhibit the proliferation of MCF-7 and MCF-7/ADM cells through inducing apoptosis and necrosis. The anti-proliferative effect of AEP on MCF-7/ADM cells was more powerful than that on MCF-7 cells(P<0.01). The mechanism of inhibition of MCF-7/ADM cells was possibly accomplished by downregulating the expression of PKM2 and FASN.