Initial Research On The Effect Of Arsenic Trioxide On Breast Cancer Cell MDA-MB-435s

Breast cancer is the most common malignant tumor in women and there are more than 1,000,000 new cases in the world every year. The incidence rate of breast cancer increases year by year in our country, and also assumes the young tendency. Breast cancer has become the significant threat to the feminine health in our country. Arsenic trioxide (As₂O₃) is a kind of traditional Chinese medicine in our country, and several centuries ago it was used by people to treat the Psoriasis, the rheumatism, the asthma, the syphilis, the hemorrhoids and so on. Since the beginning of the 70's, after As₂O₃ was successfully used in the treatment of the conventional chemotherapy or the recurring APL ( acute promyelocytic leukemia ) after the all-trans retinal treatment, Arsenic trioxide has become the new international hot spot in the research of hematology and oncology. Recent research has confirmed that As₂O₃ has obvious effects of inhibiting growth, inducing apoptosis on many kinds of malignant blood cells besides APL and solid tumor cells including liver cancer cell, renal cancer cell, pancreatic cancer cell, esophageal cancer cell, prostatic cancer cell and so on. But there is little research about the use of As₂O₃ in breast cancer now. About the anti-tumor molecular mechanism of As₂O₃, it is not really clear. Researches have found that the function mechanism of As₂O₃ is complicated, and the function ways vary with different concentration of As₂O₃ under different conditions for different types of cells. Therefore it is extremely necessary to discuss its new anti- tumor spectrum and the function mechanism.In this experiment, cell culture is used to observe the action of As₂O₃ on the carcinoma cell line MDA-MB-435s and the effect of As₂O₃ on bcl-2 and bax expression, and the possible function mechanisms are initially discussed. There are few reports about this both here and abroad. This will provide experimental basis for further application to the zooscopy of this cell and possible clinical application in the future.Objective Observe the inhibition of As₂O₃ with different concentration on MDA-MB-435s cell growth, the inducing apoptosis function on the cell and the effect on bcl-2 and bax expression, and initially discuss the mechanism of As₂O₃ inducing apoptosis.Materials and MethodsMTT assay was used to observe inhibitory effect of As₂O₃ on proliferation of MDA-MB-435s cells; The morphologic changes were studied after treatment with different concentration As₂O₃ in vitro by inverted microscope; The apoptosis was detected by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) and DNA Gel electrophoresis methods; The apoptosis-associated proteins, bcl-2 and bax, were measured by immunocytochemical technique. Statistics analysis: Use the SPSS11.0 statistical software package to carry on statistical processing. Use the single factor analysis of variance to compare any two above, and take a=0.05 as the significance level.Results1.The inhibition of As₂O₃ on the growth multiplication of the MDA-MB-435s cell in vitro.As₂O₃ of different concentration could inhibit the growth of the breast cancer cell line MDA-MB-435s, and its inhibition strengthened along with the dosage increasing, assuming the dose dependence. For 0.5μmol/ L group, 1.0μmol/ L group, 1.5μmol/ L group the growth inhibition rates were not obviously statistically different(P>0.05); between 2.5μmol/ L group and 5.0μmol/L group their growth inhibition rates were not obviously statistically different (P>0.05); between 7.5μmol/ L and 10.0μmol/ L group their growth inhibition rates were not obviously statistically different (P>0.05); and among 3 concentration gradients the growth inhibition rates were obviously statistically different between any two of them (P<0.05).2. The examination of As₂O₃ inducing MDA-MB-435s apoptosis.1) The change of cell morphology of MDA-MB-435s after the treatment with As₂O₃.Under observation by inverted microscope. The control MDA-MB-435s cell assumed the shape of shuttle or tall and slender shuttle, growing with adherence, homogeneous transparent endochylema, and good refraction; for 1.5μmol/ L group, the adherence MDA-MB-435s cells decreased, there appeared floating cells, some cells lost the normal shape, the refraction weakened, and in cell appeared the non-homogeneous granulation; for 5.0μmol/ L group there was a massive reduction of the adherence cells, the cells which felt off increased, most cells became round or small, the cell body crimpled distorted, in the cell the grana increased, nuclear chromatin pyknosised and gathered at the edge of the nuclear membrane ; for 10.0μmol/ L group the cells were sparsate and separated, most cells felt off, the cell body crimpled and was small, and the nuclear chromatin pyknosised and gathered at the edge the nuclear membrane, or the cell debris was obviously seen.2) The examination of the cell apoptosis with DNA agarose gel electrophoresis.The comparisons for the dose groups: No DNA ladder in the 1.5μmol/ L group, obvious DNA ladder appeared in 2.5μmol/ L group, 5.0μmol/ L group, 7.5μmol/ L group.3) Examining the apoptosis with TUNEL.Under the observation by optical microscope :No cell was colored brown yellow in the blank control; for the negative group there were only very few cells whose nucleus were colored brown yellow ; for the 1.5μmol/ L group, there were brown yellow colored areas and even densely brown yellow colored areas in some cell nucleus; for the 5.0μmol/ L group, there were brown yellow granas or densely brown yellow colored areas in most cell nucleus ; for 10μmol/ L group, the densely brown yellow granules appeared in most cell nucleus, hence there were apoptotic bodies.3. The effect of As₂O₃ on the apoptosis-related protein expression.bcl-2 and bax positive expressions were dark brown and they both lied in endochylema. Comparing any bcl-2 group with the negative control, there was a significant difference between them (P<0.05); comparing any two of the bcl-2 groups, there was a significant difference between them(P<0.05). There was a significant difference between any bax group and the negative control (P<0.05); the comparison between any two groups also had the statistical difference(P<0.05).Conclusions1. As₂O₃ can exert obvious inhibitory effect on MDA-MB-435s cell growth in vitro. The inhibitory effect increases with dosage escalation and shows dosage dependence.2. As₂O₃ can induce the MDA-MB-435s cell apoptosis, and the apoptosis effect and the dosage assume the positive correlation. 3.As₂O₃ regulating bcl-2 expression down and bax expression up, is one of the mechanisms of its inducing MDA-MB-435s cell apoptosis.