The antiproliferative effect of CP-P evaluated against MCF-7 and MDA-MB-231 human breast cancer cells.

(A) In vitro cytotoxicity of CP-P, with or without CYC, upon MCF-7 and MDA-MB-231 cells at different doses (800–12.5 µg/ml). The CP-P alone, or when combined with CYC, leads to significant (*P<0.01) reduction in cell proliferation even at 12.5 and 50 µg/ml doses versus untreated control. The MCF-7 cells were more sensitive against the combination treatment. Values are mean ± SD of three replicates. Untreated MCF-7 cells served as a control, CP-P treatment evidenced cell death (CD) at 100 µg/ml concentrations on MDA-MB-231 cells. The CP-P alone, or in combination with CYC, caused the most significant (*P<0.01) leakage of LDH. The combination treatment induced more cell-death and lytic effects in both cell lines than CP-P or CYC alone. (B) Light micrographs showing the MCF-7 cell morphology before and after treatment. (i) There was no effect on untreated MCF-7 cells as a control, (ii) protein (CP-P) or drug (CYC) alone treated cells induced severe cell membrane damage and inhibited MCF-7 cell growth, (iv) We also observed that MCF-7 cells treated with CP-P in combination with CYC showed remarkable morphological changes like cell shrinkage, formation of membrane blebs, nuclear and cytoplasmic condensation when observed under microscope (Symbol denotes: NC-Normal Control, MC-morphological changes, CD-Cell Death, MD - membrane damage). (C) Electron micrographs of MCF-7 cells exposed to CP-P alone, or with CYC, showing the cellular and morphological changes. The transmission electron microscopy (TEM) analysis was performed on MCF-7 human breast cancer cells exposed to the CP-P alone, or with CYC treatment to observe morphological changes. Electron micrographs of MCF-7 cells, (i) untreated control MCF-7 cells which showed normal morphology. (ii) Calotropis procera protein (CP-P) treatment exhibited cell death at 100 µg/ml concentrations, a portion of the nucleus dismantled within the cytoplasm and a large number of vacuoles were clearly visible in proteins treated cells. (iii) CYC alone treatment caused blebbing as revealed by the presence of large vacuoles on MCF-7 treated cells. (iv) These results further confirmed that the combination treatment of CP-P+CYC induce MCF-7 cells death at 12.5 µg/ml doses. MCF-7 cells treated with CP-P+CYC showed apoptotic morphology including chromatin condensation into dense granules or blocks. The cells showed loss of microvilli and blebbing of cell membrane which are characteristics of cells undergoing apoptosis.