| OBJECTIVE The obejective of this article was to study the effects of apigenin on human breast cancer and mechanism involved and then provide experimental evidence for clinic treatment of breast cancer by apigenin.METHODS1 In vitroCultured human breast cancer cell line ZR-75-30 was treated with apigenin. The changes of cell growth, proliferation and cell cycle were measured by MTT and FCM. Effects of apigenin on ability of tumors cell penetrating artificial basic membrane –Matrigel and inducing angiogenesis were recorded; Transcription of MMP-9 and Ki67 mRNA was measured by RT-PCR; Expression of bcl-2 and MMP-9 was detected by immunohistochemistry; Effect of apigenin on apoptosis was detected by TUNEL.2 In vivoZR-75-30 cells were implanted into BALB/c nude mice (nu/nu), 54 nude mice with implanted tumors were divided into 3 groups: group A (apigenin treatment), group B (TAM treatment) and group C (no treatment, control). Difference of volume and weight in animals of these groups was compared; HE staining showed histopathology changes of tumor in animals of different group; Transmission electronic microscope was used to observe the ultrastructure of tumor cells; Apoptosis of tumor cells was measured by TUNEL one week following treatment; Expression of bcl-2, MMP-9, CD34, VEGF, BRCA1 was detected by immunohistochemistry staining; Ki67 and bcl-2 protein were detected by Westernblot.RESULTS1 in vitroThe result of MTT assay showed, following treatment with apigenin in concentration of 5, 10, 20, 40, 60 and 80μmol/L, the growth inhibition rate of breast cancer cell line was 8.55%, 32.57%, 44.74%, 67.43%, 73.36% , 92.11%, respectively.FCM indicated that G2/M phase distribution increased with the increase of drug concentration.Apigenin reduced the ability of breast cancer cells penetrating artificial basic membrane and inducing angiogenesis (P<0.05), the effect of apigenin was dose-dependanted.RT-PCR indicated MMP-9 and Ki67 mRNA over expressed in breast cancer cells in control, and the expression was downregulated after apigenin treatment (P<0.05).Immunohistochemistry staining showed overexpression of bcl-2 and MMP-9 in breast cancer cells was downregulated by apigenin.TUNEL result indicated 42.3% tumor cells had the characteristics of apoptosis after apigenin treatment, while no apoptosis was found in breast cancer cells before treatment.2 in vivoGeneral condition: 10days after ZR-75-30 cell implantation, 52 of 54 animals were found injected location tumor. Successful rate of implantation was 96%. Tumors were nodular or lobulated. In some cases, ulcers were found on tumor surface. The surface of tumor was surrouded by vessels. Under light microscope, implanted tumors of group A showed degeneration and necrosis in different grade. Majority of tumor cells showed the signs of swelling, karyopyknosis, karyorrhexis or karyolysis. The number of vessels in interstitial space reduced, thrombosis was found in some vessels. In areas away from blood vessel, big lump of necrosis cells could be seen. Tumor cells of group B were only found swelling and degeneration. Tumor cells of group C maintained those characteristics when tumor cells were cultured in vitro. Transmission electronic microscopy result: tumor cells of group A showed the signs of apoptosis, karyopyknosis, karyolysis, electronic density of plasma increased and apoptosis body. Meanwhile, a lot of tumor cells swelled, necrosed, cell nuclear swelled and lysed, electronic density decreased, heterochromatin aggregated, mitochondrials swelled, intact plasma membrane was destroyed. Group B, plenty of apoptosis cells were found, tumor cells showed heterchromatin condensed in nuclear, apoptosis body appeared. Mitochondrials swelled in degree from slight to medium. Nuclears were bigger, nucleolus maginated, the numbeer of organelles decreased. Group C: breast cancer cells constructed adenic cavity-like structure, defomed and big nuclears were seen but apoptosis tumor cells were rare.T...
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