Berberine targets KIF20A and CCNE2 to inhibit the progression of nonsmall cell lung cancer via the PI3K/AKT pathway.

BACKGROUND: Nonsmall cell lung cancer (NSCLC) is the main type of lung cancer, accounting for approximately 85%. Berberine (BBR), a commonly used traditional Chinese medicine, has been reported to exhibit a potential antitumor effect in various cancers. In this research, we explored the function of BBR and its underlying mechanisms in the development of NSCLC. METHODS: Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), colony formation assays, flow cytometry, and transwell invasion assay were employed to determine cell growth, the apoptotic rate, cell invasion of NSCLC cells, respectively. Western blot was applied for detecting the protein expression of c-Myc, matrix metalloprotease 9 (MMP9), kinesin family member 20A (KIF20A), cyclin E2 (CCNE2), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway-related proteins. Glycolysis was evaluated by detecting glucose consumption, lactate production, and adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio with the matched kits. Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to analyze the level of KIF20A and CCNE2. Tumor model was established to evaluate the function of BBR on tumor growth in NSCLC in vivo. In addition, immunohistochemistry assay was employed to detect the level of KIF20A, CCNE2, c-Myc, and MMP9 in mice tissues. RESULTS: BBR exhibited suppressive effects on the progression of NSCLC, as evidenced by inhibiting cell growth, invasion, glycolysis, and facilitating cell apoptosis in H1299 and A549 cells. KIF20A and CCNE2 were upregulated in NSCLC tissues and cells. Moreover, BBR treatment significantly decreased the expression of KIF20A and CCNE2. KIF20A or CCNE2 downregulation could repress cell proliferation, invasion, glycolysis, and induce cell apoptosis in both H1299 and A549 cells. The inhibition effects of BBR treatment on cell proliferation, invasion, glycolysis, and promotion effect on cell apoptosis were rescued by KIF20A or CCNE2 overexpression in NSCLC cells. The inactivation of PI3K/AKT pathway caused by BBR treatment in H1299 and A549 cells was restored by KIF20A or CCNE2 upregulation. In vivo experiments also demonstrated that BBR treatment could repress tumor growth by regulating KIF20A and CCNE2 and inactivating the PI3K/AKT pathway. CONCLUSION: BBR treatment showed the suppressive impact on the progression of NSCLC by targeting KIF20A and CCNE2, thereby inhibiting the activation of the PI3K/AKT pathway.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. MATERIALS AND METHODS: We analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. RESULTS: LMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 µg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. CONCLUSION: LMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

The role of NAD(P)H:quinone oxidoreductase in quinone-mediated p21 induction in human colon carcinoma cells.

This study examines the role of NAD(P)H:quinone acceptor oxidoreductase (NQOR) (EC 1.6.99.2) in the metabolism of aziridinylbenzoquinones and the ensuing formation of reactive oxygen species in the induction of the cell cycle inhibitor p21 (WAF1, Cip1, or sdi1) in human colon carcinoma cells. The aziridinylbenzoquinones used were 2,5-diaziridinyl-1,4-benzoquinone (DZQ) and 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ). The cell lines used in this study, BE and HT29 human colon carcinoma cell lines, are devoid of and overexpress NQOR activity, respectively. The rate of reduction of the above quinones in BE cells proceeded at similar rates (approximately 170 nmol/min/ mg protein) and, expectedly, it was not affected by the NQOR inhibitor, dicumarol. The metabolism of DZQ in HT29 cells was largely accomplished by NQOR (approximately 94%), whereas that of AZQ was accomplished by dicumarol-insensitive reductases. The metabolism of DZQ in HT29 cells was accompanied by H2O2 formation, which was approximately 10-fold higher than that ensuing from the activation of AZQ. In agreement with these data, the production of H2O2 during the activation of DZQ by purified NQOR was approximately 10-fold higher than that of AZQ. The formation of H2O2 during the metabolism of aziridinylbenzoquinones in BE cells was 24- to 57-fold lower than that in HT29 cells. At variance with HT29 cells, H2O2 formation by BE cells was insensitive to the catalase inhibitor sodium azide. The bioactivation of AZQ and DZQ in BE cells yielded O2.- and HO. as detected by spin trapping/EPR, the intensity of the former adduct being approximately 2-fold higher than that of the latter. These signals were insensitive to dicumarol. The metabolism of DZQ in HT29 cells yielded mainly HO. and a modest contribution of O2.- (ratio HO./O2.- approximately 10), whereas that of AZQ yielded a HO./O2.- approximately 2. The effect of dicumarol on the free radical pattern obtained during DZQ metabolism resulted in a strong inhibition (80%) of HO. production and a substantial increase of O2.- generation. The metabolism of DZQ and AZQ in BE cells was associated with a significant increase of p21 mRNA levels; the former quinone was approximately 2-fold more efficient than the latter. DZQ metabolism in HT29 cells led to an increase of p21 mRNA levels 15-fold higher than that observed with AZQ activation. Dicumarol did not inhibit p21 induction associated with the metabolism of DZQ in the NQOR-deficient BE cells, whereas the inhibitor decreased p21 induction in HT29 cells by approximately 30%. This modest inhibition is likely due to the low concentration of dicumarol used, which did not affect p21 constitutive levels in control experiments carried out in the absence of the quinone. p21 induction in HT29 cells was also inhibited by DTPA, a metal chelator, and N-acetylcysteine, a potent cellular anti-oxidant, suggesting that HO. may serve as an ultimate mediator for the induction. It may be surmised that the higher efficiency of DZQ in p21 induction may be related to its efficient metabolism by NQOR in HT29 cells and the associated high level of reactive oxygen species. The role of reactive oxygen species in p21 induction was further assessed upon supplementation of cells with H2O2:p21 induction in BE cells was 4-fold higher than that in HT29 cells. These findings suggest that assessment of the role of NQOR and reactive oxygen species in p21 induction requires careful consideration of the cell genotype.