BackgroundThe extracts from Artemisia annua Linné (AAE) has been known to possess various functions including anti-bacterial, anti-virus and anti-oxidant effects. However, the mechanism of those effects of AAE is not well known. Pursuantly, we determined the apoptotic effects of extract of AAE in HCT116 cell. In this study, we suggested that AAE may exert cancer cell apoptosis through PTEN/PDK1/Akt/p53signal pathway and mitochondria-mediated apoptotic proteins.MethodsWe measured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) assay, Hoechst 33342 staining, Annexin V-PI staining, Mitopotential assay, immunofluorescence (IF) and Western blotting. Accordingly, our study showed that AAE treatment to HCT116 cells resulted in inhibition of PDK1, Akt, MDM2, Bcl-2, and pro-caspase 3 as well as activation of PTEN, p53-upregulated modulator of apoptosis (PUMA), Bax and Bak expression. Also we measured in vivo assay that xenograft model, H&E assay, TUNEL assay and IHC.ResultsAAE induced apoptosis via PTEN/p53/PDK1/Akt signal pathways through PTEN/p53-independent manner. AAE inhibit cell viability and increase LDH release in HCT116 colon cancer cell. Also, AAE increase apoptotic bodies, caspase −3,7 activation and reduces mitochondria membrane potential. AAE regulates cytochrome c translocation to the cytoplasm and Bax translocation to the mitochondrial membrane in an Immunofluorescence staining and increase PTEN and p53 expression in an in vivo tumor xenograft model. To elucidate the role of the PTEN/p53/PDK1/Akt signal pathways in cancer control, we conditionally inactivated PTEN/p53/PDK1/Akt signal pathways. We used inhibitors of PTEN, p53, PDK1, Akt. In consequence, these results indicate that AAE induced apoptosis by means of a mitochondrial event through the regulation of proteins such as Bax, Bak and cytochrome c in PDK1/Akt signaling pathways via PTEM/p53-independent manner.ConclusionsWe confirmed the apoptotic effect of extracts of AAE by Modulating PTEN/p53/PDK1/Akt/Signal Pathways through PTEN/p53-independent pathwaysin HCT116 colon cancer cell.

Cnidium monnieri (L.) Cusson is a frequently used traditional Chinese medicine that treats gynecological diseases and carbuncles. However, the mechanism of action of C. monnieri remains to be fully elucidated. The present study examined the cell cycle arrest and apoptotic effects resulting from ethanol extract of C. monnieri (CME) in HepG2 (wild‑type p53) and Hep3B (p53‑null) hepatocellular carcinoma cells. An MTT assay was used to confirm the anti‑proliferative effect of CME. The cells were stained with Hoechst 33342 or propidium iodide. It was demonstrated that proliferation of HepG2 cells was suppressed by CME. Cell cycle arrest occurred in the G1 phase following treatment with CME and the number of apoptotic bodies was increased. The expression levels of cell cycle‑associated proteins, including protein kinase B (Akt), glycogen synthase kinase‑3β (GSK‑3β), p53, cyclin E and cyclin‑dependent kinase 2 (CDK2) were determined by western blot analysis. The protein levels of phosphorylated (p)‑Akt, p‑GSK‑3β, p‑MDM2 and cyclin E were decreased, whereas the protein levels of p53, p21 and p‑CDK2 (Thr14/Tyr15) were increased following treatment with CME. Furthermore, treatment or co‑treatment with LY294002 (phosphoinositide‑3‑kinase/Akt inhibitor) or Pifithrin‑α (p53 inhibitor) with CME resulted in CME‑induced G1 arrest which occurred through the p53‑independent signaling pathway in hepatocellular carcinoma cells. In conclusion, CME induces G1 arrest and apoptosis via the Akt/GSK‑3β signaling pathway which is regulated by MDM2‑induced degradation of p21, rather than p53.

Colon cancer, a common malignancy, can occur due to poor eating habits and increasing age. Consequently, careful regulation of eating habits may serve as a possible method for preventing the occurrence or progression of colon cancer. Extracts of the fruit of Cnidium monnieri (L.) Cusson are well‑known as an effective herbal medicine for the treatment of pain in female genitalia and carbuncle. However, there have been no studies on the apoptotic effects of Cnidium monnieri (L.) Cusson (CME). Adenosine monophosphate‑activated protein kinase (AMPK), the major regulator of energy metabolism, is activated by metabolic stress, including hypoxia and glucose deprivation. Activation of AMPK inhibits cell proliferation and induces apoptosis through the inhibition of phosphorylated (p)‑Akt and control of B‑cell lymphoma 2 (Bcl‑2) family members. The pro‑apoptotic proteins Bcl‑2‑associated X protein (Bax) and Bcl‑2‑homologous antagonist killer (Bak), are activated by their translocation to mitochondria from the cytosol. Translocation of Bax/Bak induces outer membrane permeabilization and is likely to lead to apoptosis through cytochrome C release and caspase activity. In the present study, the apoptotic effects and influence on mitochondria‑mediated apoptotic proteins of CME in HCT116 cells were assessed. We hypothesized that CME may have an effect on the inhibition of p‑Akt in an AMPK‑independent pathway. The present study demonstrated that CME induced the release of LDH and apoptosis through its inhibition of p‑Akt to control Bcl‑2 and activate Bax and Bak. Co‑treatment with CME and AMPK inhibitors showed that CME‑induced apoptosis does not occurr through a AMPK‑dependent pathway. Therefore, the present study determined, for the first time, that CME induced apoptosis as a result of causing metabolic stresses due to directly regulation of the de‑phosphorylation of Akt, whereas it did not control the AMPK-dependent pathway in HCT116 colon cancer cells.

In this study, extract from Artemisia annua in L. (AAE) is known as a medicinal herb that is effective against cancer. The cell cycle is regulated by the activation of cyclindependent kinase (CDK)/cyclin complex. We will focus on regulation of CDK2 by cyclin E. cyclin E is associated with CDK2 to regulate progression from G1 into S phase. Akt is known to play an important role in cell proliferation and cell survival. Activation of Akt increases mTOR activity that promotes cell proliferation and cancer growth. In this study, we investigated that AAE-induced cell cycle arrest at G1/S phase in HCT116 colon cancer. Treatment of AAE shows that reduced activation of Akt decreases mTOR/Mdm2 activity and then leads to increase the activation of p53. The active p53 promotes activation of p21. p21 induces inactivation of CDK2/ cyclin E complex and occurs cell cycle arrest at G1/S phase. We treated LY294002 (Akt inhibitor) and Rapamycin (mTOR inhibitor) to know the relationship between the signal transduction of proteins associated with cell cycle arrest. These results suggest that AAE induces cell cycle arrest at G1/S phase by Akt/mTOR pathway in HCT116 colon cancer cell.

No abstract