Calotropis gigantea stem bark extract induced apoptosis related to ROS and ATP production in colon cancer cells

Abstract

Conventional chemotherapeutic agents for colorectal cancer (CRC) cause systemic side effects and eventually become less efficacious owing to the development of drug resistance in cancer cells. Therefore, new therapeutic regimens have focused on the use of natural products. The anticancer activity of several parts of Calotropis gigantea has been reported; however, the effects of its stem bark extract on inhibition of cancer cell proliferation have not yet been examined. In this study, the anticancer activity of C. gigantea stem bark extract, both alone and in combination with 5-fluorouracil (5-FU), was evaluated. A crude ethanolic extract was prepared from dry, powdered C. gigantea barks using 95% ethanol. This was then partitioned to obtain dichloromethane (CGDCM), ethyl acetate, and water fractions. Quantitative analysis of the constituent secondary metabolites and calotropin was performed. These fractions exhibited cytotoxicity in HCT116 and HT-29 cells, with CGDCM showing the highest potency in both the cell lines. A combination of CGDCM and 5-FU significantly enhanced the cytotoxic effect. Moreover, the resistance of normal fibroblast, HFF-1, cells to this combination demonstrated its safety in normal cells. The combination significantly enhanced apoptosis through the mitochondria-dependent pathway. Additionally, the combination reduced adenosine triphosphate production and increased the production of reactive oxygen species, demonstrating the mechanisms involved in the induction of apoptosis. Our results suggest that CGDCM is a promising anti-cancer agent and may enhance apoptosis induction by 5-FU in the treatment of CRC, while minimizing toxicity toward healthy cells.

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine

“…This may be due to the distribution of phenolic compounds to other fractions by subsequent liquid–liquid partition. However, these results showed the same trend as a previous report 66 . Among the tested samples, CGDCM comprised the highest amounts of triterpenoids, flavonoids and calactin, while CGEtOAc contained the highest contents of cardiac glycosides and phenolic compounds.…”

“…Our present study indicated that all four tested samples, including CGEtOH, CGDCM, CGEtOAc and CGW, contained cardiac glycosides, triterpenoids, phenolic compounds, flavonoids, alkaloids and calactin with different magnitudes similar to those in a previous report by our group 66 . The total contents of phenolic compounds and flavonoids reported in this study corresponded to those found in C. gigantea flowers 16 .…”

“…The results revealed that the percent yield of CGEtOH (3.3%) obtained from the maceration method at room temperature (27 ± 5 °C) was less than that in our previous report (5.6%) 66 using ultrasonic-assisted extraction. CGEtOH was fractionated to divide the phytochemicals according to their solubilities in each organic solvent.…”

Reactive Oxygen Species: Not Omnipresent but Important in Many Locations

“…This further places the model of ROS molecules, which “flood” the whole cell from a single location of ROS production in rather unrealistic light. Nevertheless, the model of ROS as “flooding” the cell is still popular in the scientific community, often referred to as “total cellular ROS levels” ( Wang et al, 2013 , 2019 ; Dinakar et al, 2016 ; Kim and Xue, 2020 ; Loth et al, 2020 ; Thorne et al, 2021 ), “intracellular ROS levels” ( Tepel et al, 2000 ; Bensaad et al, 2009 ; Choi et al, 2011 ; Lee et al, 2017 ; Wang et al, 2019 , 2021a ; Wei et al, 2019 ; Zaidieh et al, 2019 ; Zhang W. et al, 2019 ; Mendiola et al, 2020 ; Winitchaikul et al, 2021 ; Zhong et al, 2021 ) or simply “ROS levels” ( Chen et al, 2012 , 2021 ; Wei et al, 2019 ; Agarwal and Ganesh, 2020 ; Kim et al, 2021 ; Knight et al, 2021 ; Zeller et al, 2021 ) in many studies, mainly because of the usage of diffusible ROS probes, which suggest free diffusion of ROS through the cell without regard of the location of ROS production. Mutations, e.g., in cancer cells ( Schumacker, 2006 ; Liou and Storz, 2010 ; Reczek and Chandel, 2017 ; Zaidieh et al, 2019 ; Perillo et al, 2020 ), pathogenic invasion ( West et al, 2011a ; Abuaita et al, 2018 ; Gluschko et al, 2018 ; Roca et al, 2019 ) or metabolic disbalance ( Li et al, 2016 ; Mak et al, 2017 ; Peng et al, 2021 ) are prominent examples, in which the ROS production of the cell can enter an uncontrolled stage and quickly overcome the antioxidative defense system leading to rapidly increased ROS levels in nearly every compartment of the cell with often detrimental consequences.…”

An updated pharmacological insight into calotropin as a potential therapeutic agent in cancer

“…The combination of low concentrations of 5-FU and calotropin in comparison to a high dose of 5-FU reduced cell viability and abolish resistance to this conventional chemotherapeutic in HCT116 colorectal cancer cells. The same treatment significantly reduced the cellular ATP levels and increase ROS levels in HCT116 cells ( Winitchaikul et al, 2021 ). Additionally, the increased intracellular concentration of ROS triggers autophagy and induced apoptotic cell death suppressing the activity of antioxidative enzymes ( Scherz-Shouval et al, 2019 ).…”