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It considered to be an appealing therapeutic target. Makinoshima et al. And mTOR inhibition contributes to the initiation of autophagy even in the presence of sufficient nutrients and growth hormones.
Cardamonin appears to have anti-inflammatory, antineoplastic, vasorelaxant and anti-infectious properties which benefit for the potential health applications [ 12 ]. Cardamonin inhibits the proliferation and metastasis of various cancer cells [ 13 , 14 , 15 ]. In our previous studies, we demonstrate that the antitumor effect of cardamonin dependents on mTOR inhibition [ 16 , 17 ]. Recently, we find that the autophagy induction is account for the antiproliferative activity of cardamonin [ 18 ].
Also, cardamonin plays an important role in glucose metabolism. In the present study, we aim to explore the mechanism of cardamonin on autophagy. It will provide a promising therapeutic agent for ovarian cancer. The content of lactate was normalized to cell numbers. We have investigated the antiproliferation effect of cardamonin in various cancer cells.
Treated with indicated drugs as described above, the cells were washed twice with ice cold PBS and harvested with 0. The cell suspension was washed with ice cold PBS for three times. The protein content was measured by the BCA assay kit. The absorbance was measured again under the same conditions and denoted as OD2. At last 2. Cells were cultured in 6-well plates 0. Then the cells were washed twice with PBS and observed by fluorescence microscope.
The protein concentration was measured by a BCA assay kit. Glycolysis is characterized with the conversion of glucose into lactate [ 20 ]. We measured the content of lactate to assess the inhibitory effect of cardamonin on glycolysis. As shown in Fig. Cardamonin inhibited lactate secretion.
Lactate secretion was analyzed by an assay kit. Treated with cardamonin and rapamycin, the fluorescent intensity of MDC stain was significantly elevated Fig. As a glycolysis inhibitor, 2-DG also induced autophagy Fig.
Cardamonin induced cell autophagy. This result was in accordance with the glycolysis inhibition described above Fig. Next we investigated whether cardamonin-induced autophagy was correlated with glycolysis inhibition. The relationship between the activity of AMPK and autophagy by cardamonin. Recently, Muilenburg et al. Dasatinib can induce cell death through autophagy in ovarian cancer [ 22 ].
In addition, increased expression of autophagy protein PEAprolonged the life of ovarian cancer patients [ 23 ]. Therefore, autophagy provides new ideas for the treatment of ovarian cancer. LC3 is essential for the elongation of autophagosome membrane. LAMP1 is a structural protein of the lysosome membrane, which is also considered as an indicator for autophagy [ 24 ].
These effects of cardamonin were consistent with that of other similar flavonoids, such as genistein and baicalein [ 25 , 26 ]. The mechanism of rapamycin and 2-DG on autophagy induction has been demonstrated in other studies. When nutrients are deprived, autophagy is enabled by impaired cellular ATP production. As the key point of energy metabolism, glycometabolism plays a unique role in maintaining ovarian cancer cell growth [ 29 , 30 , 31 ].
HK is involved in the glycolysis pathway in cancer cells [ 32 ]. HK catalyzes the first step of glycolysis. It phosphorylates glucose to glucosephosphate. HK2 is a predominant isoform of HK, which is upregulated in ovarian cancer [ 33 ]. LDH sustains the glycolysis through catalyzing the regeneration of nicotinamide adenine dinucleotide from reduced nicotinamide adenine dinucleotide in cancer cells [ 35 ]. It demonstrates that various flavonoids, including luteolin, wogonin, etc.
It contributes to the glycolysis inhibitory effect of cardamonin. The results were similar with 2-DG, a glucose analog that competitively phosphorylated by HK with glucose and further inhibits glycolysis [ 28 , 38 ]. It indicated that cardamonin inhibits glycolysis induced energy production. Interestingly, we found that both cardamonin and 2-DG decreased the relative protein expression of HK2.
However, only cardamonin but not 2-DG inhibited the activity of HK. The underling mechanism needs to be further illustrated. It demonstrates that mTORC1 inactivation by rapamycin is critical for glucose metabolism [ 41 , 42 ]. Our previous study showed that cardamonin had a similar effect with rapamycin on mTOR and its downstream substrates, but this effect was not dependent on FKBP12 [ 17 ]. Therefore, it suggested that the inhibition of glycolysis by cardamonin was regulated by mTORC1 suppression.
AMPK plays an important role in adaptive responses to reduced energy production. In order to clarify the activation of AMPK partly results in autophagy induction, we added the Compound C in the experiment. Cardamonin-induced autophagy was reduced by Compound C. All these findings suggested that autophagy stimulation by cardamonin was involved in glycolysis inhibition mediated AMPK activation. Cardamonin exhibits antiproliferation activity in various cancer cells.
This study also demonstrates that cardamonin-induced autophagy is associated with glycolysis inhibition via mTOR inhibition in SKOV3 cells. Our increased understanding about the mechanism of antitumor effect of cardamonin provides important evidences for further development of cardamonin as a novel therapeutic drug for ovarian cancer.
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