Chloroxylon swietenia (Roxb.) DC induces cell death and apoptosis by down‐regulating the NF‐κB pathway in MCF‐7 breast cancer cells: In vitro and in vivo investigations

Abstract

Background Natural products with targeted bioactivity have gained major attention in the field of cancer research owing to emerging anti‐cancer drug resistance and off target toxicities. Chloroxylon swietenia (Roxb.) DC is recognized as a folklore medicinal plant and has numerous therapeutic benefits in the folklore medicine system, however the anti‐cancer potential of this plant and its mechanism of action is poorly understood. Aims The aim of the study was to investigate the anti‐breast cancer efficacy of C. swietenia leaves methanol extract (CSLME) against MCF‐7 hormone dependent human breast cancer cell line with possible mechanism of action. Methods and results The anti‐breast cancer activity of CSLME against MCF‐7 cells was assessed by evaluating its efficacy toward cytotoxicity, cell migration, colony formation, DNA fragmentation, apoptosis, cytoskeleton, angiogenesis, cell cycle regulation, and animal toxicity. The preliminary screening of CSLME against MCF‐7 cells revealed the cytotoxicity (IC 50 20 μg/ml), inhibited cell migration, colony formation, and angiogenesis. It was observed that CSLME induces apoptosis by nuclear fragmentation and disruption of cytoskeleton by actin derangement. The results of Annexin V‐FITC assay and cell cycle analysis by flow cytometry clearly pointed out the sizable fraction of apoptotic cells, and arrested the cells at G2/M phase of cell cycle. The results of the immunoblotting experiments showed that CSLME activates intrinsic pathway of apoptosis with down regulation of anti‐apoptotic marker like Bcl2, up regulation of pro‐apoptotic markers like Bax & Bad, along with successful cleavage of Caspase‐9 and PARP‐1. Further, western blot analysis revealed the possible down regulation of NF‐κB pathway by CSLME, which may be responsible for anti‐cancer activity in MCF‐7 cells. In vivo animal model studies using NOD‐SCID mice demonstrated impressive anti‐tumor activity with significant reduction in tumor volume of MCF‐7 tumor xenograft. Of note, in‐vivo acute oral toxicity study as per Organization for Economic Cooperation and Development 423 revealed the nontoxic nature of CSLME. Conclusion The in vitro and in vivo findings clearly outline the potential of CSLME as inhibitor of growth and proliferation of MCF‐7 cells. Mechanistically, CSLME seems to activate intrinsic pathway of apoptosis, arrest cell cycle, target actin cytoskeleton, inhibit growth, colony formation, migration, and angiogenesis, with down regulation of NF‐κB pathway leading to cell death.

Repurposing of neprilysin inhibitor ‘sacubitrilat’ as an anti-cancer drug by modulating epigenetic and apoptotic regulators

“…The effect of sacubitrilat and SAHA (reference drug) on the cell viability of colon and TNBC cancer cells was assessed using an MTT cell proliferation assay. The MTT assay protocol was adopted from earlier studies 71 – 73 . In brief, the SW-480 and MDA-MB-231 cells were seeded at a cell density of 1 × 10 3 cells/per well into a 96-well culture plate.…”

“…The scratch assay was performed as described in our earlier reports 72 , 73 . Briefly, the colon cancer cells were seeded in 24 well plates (1 × 10 4 cells/well) and grown till confluency.…”

“…To investigate the effect of sacubitrilat on the regulation and phase distribution of the cell cycle in SW-480 cells, we performed propidium iodide (PI) staining using flow cytometry analysis 72 , 73 . The SW-480 (1 × 10 6 ) cells were seeded in 35 mm plates and treated with sacubitrilat and SAHA for 48 h. After treatment, cells were stained with PI (50 μg/mL).…”

“…To investigate the role of sacubitrilat in the induction of apoptosis in SW-480 cells, we have performed the Annexin-V-FITC assay using FACS analysis 71 – 73 . The assay was carried out using Annexin V-FITC Apoptosis Detection Kit (Biolegend).…”

“…Similarly, pro-apoptotic markers (BAX, BID and PARP) and anti-apoptotic BCL2 markers were studied in SW-480 after the treatment of sacubitrilat and SAHA. The WBA was carried out as per the previously described method 72 , 73 . In brief, the SW-480 cells (2 × 10 5 ) were seeded in 60 mm plates and treated with sacubitrilat and SAHA.…”