Abstract
BackgroundColorectal cancer has become one of the leading cause of cancer morbidity and mortality throughout world. Hederagenin, a derivative of oleanolic acid isolated from the leaves of ivy (Hedera helix L.), has been shown to have potential anti-tumor activity. The study was conducted to evaluate whether hederagenin could induce apoptosis of human colon cancer LoVo cells and explore the possible mechanism.MethodsMTT assay was used for evaluating cell viability while Annexin V-FITC/PI assay and Hoechst 33342 nuclear stainining were used for the determination of apoptosis and mitochondrial membrane potential. DCFH-DA fluorescence staining and flow cytometry were used to measure ROS generation. Real-time PCR and western blot analysis were performed for apoptosis-related protein expressions.ResultsMTT assay showed that hederagenin could significantly inhibit the viability of LoVo cells in a concentration-dependent and time-dependent manner by IC50 of 1.39 μM at 24 h and 1.17 μM at 48 h. The apoptosis ratio was significantly increased to 32.46% and 81.78% by the induction of hederagenin (1 and 2 μM) in Annexin V-FITC/PI assay. Hederagenin could also induce the nuclear changes characteristic of apoptosis by Hoechst 33342 nuclear stainining under fluorescence microscopy. DCFH-DA fluorescence staining and flow cytometry showed that hederagenin could increase significantly ROS generation in LoVo cells. Real-time PCR showed that hederagenin induced the up-regulation of Bax and down-regulation of Bcl-2, Bcl-xL and Survivin. Western blotting analysis showed that hederagenin decreased the expressions of apoptosis-associated proteins Bcl-2, procaspase-9, procaspase-3, and polyADP- ribosepolymerase (PARP) were increased, while the expressions of Bax, caspase-3, caspase-9 were increased. However, there was no significant change on caspase-8.ConclusionsThese results indicated that the disruption of mitochondrial membrane potential might contribute to the apoptosis of hederagenin in LoVo cells. Our findings suggested that hederagenin might be a promising therapeutic candidate for human colon cancer.
Highlights
Colorectal cancer has become one of the leading cause of cancer morbidity and mortality throughout world
The IC50 for hederagenin in LoVo cells were 1.39 μM at 24 h for and 1.17 μM at 48 h, respectively. These results suggested that hederagenin showed a dose-dependent and time-dependent inhibitive effect on the proliferation of LoVo cells
The characteristic features of apoptosis including chromatin condensation and nuclear fragmentation in hederagenintreated cells were observed as compared with the control cells (Figure 3B). These results demonstrated that the inhibition of hederagenin on the growth of LoVo cells was associated with its induction of apoptosis
Summary
Colorectal cancer has become one of the leading cause of cancer morbidity and mortality throughout world. Several epidemiological studies have reported that the mean cumulative incidence rate of colorectal cancer is 3.1% at 10 years [3,4]. The risk of this disease is multifactorial, such as age, environmental, genetic, and dietary factors. The etiology of colorectal cancer is limited, Currently, therapeutic methods for human colorectal cancer include radiotherapy, chemotherapy and surgery [5]. These strategies for the treatment of colorectal cancer are not satisfactory. Effective strategy needs to be developed to decrease the morbidity and mortality of colorectal cancer
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