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. 2014 Dec;21(6):582-8.
doi: 10.1016/j.sjbs.2014.03.002. Epub 2014 Mar 31.

Molecular mechanisms of leptin and pro-apoptotic signals induced by menadione in HepG2 cells

Ebtesam Al-Suhaimi  1
Affiliations

Molecular mechanisms of leptin and pro-apoptotic signals induced by menadione in HepG2 cells

Ebtesam Al-Suhaimi. Saudi J Biol Sci. 2014 Dec.

Abstract

Apoptosis is a significant physiological function in the cell. P(53) is known as tumor suppressor cellular factor, executive caspases are also the most involved pathway for apoptosis. Menadione (VK3) has apoptotic action on many harmful cells, but the molecular role of adipokines is not studied enough in this regard, so the ability of menadione to modify the adipokine (leptin hormone), caspase-3 and P(53) signals to induce its apoptotic action on HepG2 cells was studied. The study revealed that menadione has anti-viability and apoptotic effect at sub-G1 phase of HepG2 cell cycle. Its cytotoxic effect is mediated by molecular mechanisms included: inhibiting leptin expression and level, activating caspase-3 pathway and up-regulating the expression of P(53). Menadione exerts its apoptotic mechanisms in a concentration and time dependent way through ROS generation. In addition to the known apoptotic pathways, the results indicate that suppressing leptin pathway is a significant mechanism for menadione apoptotic effect which made it as a potential therapeutic vitamin in preventing hepatocyte survival and proliferation.

Keywords: Apoptosis; Caspase-3; HepG2 cells; Leptin; Menadione; P53; ROS.

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Figures

Figure 1
Figure 1
(A) HepG2 cells were incubated with different concentrations (5–40 μM) of menadione for 24 h. Cell viability was assessed by MTT assay. Data present mean ± SD, where p ⩾ 0.05. (B) HepG2 cells were incubated with menadione (10 μM) for indicated time. Cell viability was assessed by MTT assay. Data present mean ± SD, where p ⩾ 0.05.
Figure 2
Figure 2
(A) HepG2 cells were treated with 10 μM of menadione for indicated time and stained with P1 and their DNA content was analyzed by FACS. Menadione increases cell fraction at sub-G1 phase in a time dependent way, which is indicative of apoptosis. The sub-G1 apoptotic cells (%) were placed in the graph against the indicated time of 10 μM menadione effect. Data express mean ± SD at p ⩾ 0.05. (B) At the indicated time, menadione (10 μM) treatment induces the expression of P53 up-regulation in a time dependent way. β-actin level is used as a loading control.
Figure 3
Figure 3
Shows the ability of menadione to activate caspase 3 signal pathway. (A) Menadione induces caspase-3 activity in a time dependent way after incubation HepG2 cells with the enzyme substrate. Data express mean ± SD at p ⩾ 0.05. (B) At the indicated time, menadione treatment decreases procaspase-3 expression, which is an indicative for casapse-3 activation in a time-dependent manner.
Figure 4
Figure 4
Menadione inhibits leptin pathway. (A) Leptin levels in menadione treated HepG2 cell culture media were measured by ELISA and revealed that menadione (10 μM) decreases leptin levels in a time dependent way. (B) Also Western blot analysis demonstrated a decrease in leptin expression with time.
Figure 5
Figure 5
Shows that menadione (10 μM) alone induced ROS generation in HepG2 cells compared with control cells. But treating the cells with NAC followed by 10 μM menadione inhibited ROS generation.
None
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