Abstract
Purpose: To study the effect of Compound Bieshe Kang’ai (CBK) on proliferation and apoptosis in colorectal cancer cells.Methods: HCT116 colorectal cancer cells and FHs 74 Int intestinal cells were treated with CBK, followed by determination of cell proliferation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Caspase-9 and caspase-3 activities as well as protein expressions of Bcl-2 and BAX, and mRNA levels of caspase-9, caspase-3, Bcl-2 and BAX in HCT116 cells were evaluated, followed by examination of the morphological alterations of HCT116 cells with Hoechst 33342 staining.Results: CBK suppressed proliferation of HCT116 cells in a concentration- and time-dependent pattern, without cytotoxicity to FHs 74 Int cells. CBK also elevated caspase-9 and caspase-3 activities, mitigated protein translation of Bcl-2 and augmented that of BAX. It also enhanced mRNA transcriptions of caspase-9, caspase-3 and BAX, but decreased that of Bcl-2 in HCT116 cells in a concentrationdependent manner, as well as induced cancer cell shrinkage, nuclear fragmentation and chromatin condensation.Conclusion: The findings highlight CBK as a promising therapeutic agent for colorectal cancers, by retarding proliferation and inducing apoptosis in cancer cells.Keywords: Apoptosis, BAX, Bcl-2, Cancer, Caspase, Compound Bieshe Kang’ai, Chromatin condensation, Nuclear fragmentation
Highlights
Apoptosis is programmed cell death mainly via intrinsic pathway [1]
It is known that reduced apoptosis and over proliferation are associated with initiation and progression of divers cancer types, which are characterized by impaired apoptotic signaling
The study showed that Compound Bieshe Kang’ai (CBK) suppressed the proliferation in HCT116 cells in a concentration- and time-dependent manner, which coincided with other reports in which Hedyotis diffusa Willd, a Chinese herb of CBK, suppresses colorectal cancer growth through multiple cellular pathways [6], and Radix Astragali that helped carboplatin to inhibit B16 tumor cell growth [7]
Summary
Apoptosis is programmed cell death mainly via intrinsic pathway [1]. While apoptotic pathway is activated by various stimuli, family members of B-cell lymphoma 2 are down regulated, which renders pro-apoptotic members to perturb the mitochondria. HCT116 cells or FHs 74 Int cells were treated by CBK at diverse concentrations for various time intervals, followed by the tests as detailed below. After treatment of HCT116 cells or FHs 74 Int cells with CBK at different concentrations for various durations, MTT solution was aseptically added, followed by 4-hour incubation. Following treatment of HCT116 cells grown in a 6-well plate with CBK for 48 h at 125, 250 and 500 μg/mL, cells were collected for examination of caspase activity with caspase-9 assay kit and caspase-3 assay kit respectively, in accordance with the user’s manual. After 24-hour treatment with CBK at 125, 250 and 500 μg/mL, HCT116 cells were processed for RNA extraction with RNeasy mini kit, followed by reverse transcription (RT) of 500ng of RNA per sample to cDNA with reverse transcription kit.
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