ABSTRACT
Background The key enzymes of serine synthesis pathway (SSP) play an important role in tumor growth, proliferation, and invasion, but their roles in arsenic carcinogenesis are unclear. Objective To observe the effects of NaAsO2 treatment on the expressions of key enzymes [such as phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)] of SSP and on the ability to proliferate and migrate in human immortalized skin keratinocytes (HaCaT) and NaAsO2-induced malignantly transformed HaCaT (T-HaCaT), and to explore the roles of SSP key enzymes in arsenic carcinogenesis. Methods (1) The T-HaCaT cells constructed earlier by our research team were divided into a passage control (0 μmol·L−1 NaAsO2) group, a T-HaCaT (0.5 μmol·L−1 NaAsO2) group, a NCT503 (PHGDH inhibitor, 25 μmol·L−1) group, and a NCT503 (25 μmol·L−1) + T-HaCaT (0.5 μmol·L−1 NaAsO2) group. Western blotting was used to detect the protein expression levels of SSP key enzymes in the passage control group and the T-HaCaT group. CCK8 assay and cell scratch test were used to detect the proliferation and migration rates of cells in each group respectively. (2) Well-grown logarithmic-phase HaCaT cells were treated with 0, 0.625, 1.25, and 2.5 μmol·L−1 NaAsO2 for 0, 24, 48, and 72 h to detect cell proliferation rate and protein expression levels of SSP key enzymes. In the subsequent experiment, HaCaT cells were pretreated with 25 μmol·L−1 NCT503 for 6 h, and then treated with 2.5 μmol·L−1 NaAsO2 for 72 h continuously. The experimental groups included a control (0 μmol·L−1 NaAsO2) group, an exposure (2.5 μmol·L−1 NaAsO2) group, a pretreatment (25 μmol·L−1 NCT503) group, and a pretreatment (25 μmol·L−1 NCT503) + exposure (2.5 μmol·L−1 NaAsO2) group, to detect the proliferation rate of cells in each group. Results The protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P<0.05), and its proliferation rate (177.51%±14.69%) and migration rate (53.85%±0.94%) were also higher than the passage control group’s (100.00%±0.00% and 24.30%±2.26%) (both Ps<0.05), respectively. After the NCT503 intervention, the proliferation rate (144.97%±8.08%) and migration rate (35.80%±0.99%) of cells in the NCT503 + T-HaCaT group were lower than those in the T-HaCaT group (both P<0.05). The proliferation rate of HaCaT cells after NaAsO2 exposure for 72 h increased with the increase of exposure concentration (r=0.862, P<0.05), and consistently, the protein levels of SSP key enzymes in HaCaT cells in each exposure group were higher than those in the control group (all P<0.05). The proliferation rate of HaCaT cells treated with 2.5 μmol·L−1 NaAsO2 increased with the extension of exposure time (r=0.775, P<0.05), which was consistent with the changes of PHGDH levels in cells. After the NCT503 intervention, the proliferation rate of the pretreatment + exposure group was significantly lower than that of the exposure group (P<0.05). Conclusion The key enzymes of SSP may play an important role in the proliferation of T-HaCaT cells induced by NaAsO2.
ABSTRACT
OBJECTIVE：To establish a method for the content determination of apigenin and piperine in the water extract as well as eucalyptol and cumin aldehyde in the volatile oil of Mongolian medicine Sugmel- 3 decoction. METHODS ：HPLC method was adopted for the content determination of apigenin and piperine. GC method was used for the content determination of eucalyptol and cumin aldehyde. The determination of HPLC method was performed on Agilent Eclipse XDB-C 18 column with mobile phase consisted of methanol- 0.1％ phosphoric acid aqueous solution at flow rate of 1.0 mL/min；the detection wavelength was set at 225 nm（apigenin）and 342 nm（piperine）；the column temperature was set at 30 ℃ with sample size of 10 μL. The determination of GC method was performed on Dimensions SH-Rtx- 1 capillary column with high-purity hydrogen as carrier gas ； the injector temperature was set at 270 ℃，with flow rate of carrier gas 1 mL/min by temperature programmed ；the sample size was 1 μL，and split ratio was 1 ∶ 10. RESULTS：The linear ranges of apigenin ，piperine，eucalyptol and cumin aldehyde were 12.5-200 μg/g/mL（r＝0.999 6），87.3-139.7 μg/mL（r＝0.999 9），136-2 187 μg/mL（r＝0.999 9），39-635 μg/mL（r＝0.999 9）， respectively. The quantitation limits were 0.02，0.06，0.06，0.12 μg/mL，respectively. The detection limits were 0.01，0.02，0.03， 0.04 μg/mL. RSDs of precision，stability and reproducibility tests were all less than 4％. The recovery rates of the samples were 89.26％ -97.26％（RSD＝2.69％ ，n＝6），94.20％ -104.01％（RSD＝3.64％ ，n＝6），98.51％ -110.11％（RSD＝3.87％ ，n＝6）， 95.76％-107.82％（RSD＝4.12％，n＝6），respectively. The contents of above components were 0.769-0.828，7.741-7.981，5.284 7- 5.846 6，1.038 6-1.101 2 mg/g（n＝3）. CONCLUSIONS：The established method is simple and feasible ，and can be used for quality control of different parts of Mongolian medicine Sugmel- 3 decoction.