OBJECTIVEThis study aimed to induce carcinogenesis of lingual mucosa in C57BL/6 mice by feeding them 4-nitroquinoline 1-oxide (4NQO) solution.

METHODSA total of 85 C57BL/6 mice were randomly divided into distilled water control group (DD group, n=5), 1,2-propylene glycol control group (PG group, n=5), and experimental group (EP group, n= 75). The mice in the experimental group were medially fed in 15 cages. By contrast, the mice in DD, EP, and PG groups were watered with distilled water, 50 mg.L-1 4NQO solution, and 1,2-propylene glycol solution. The mice in EP group were executed every two weeks from week 0, and the mice in the control groups were sacrificed at the 28th week. The mice were weighed. Mucosal lesions were measured by macroscopic observation and histopathologic detection.

RESULTSOne mouse in EP group died of unknown reason. The weight of the mice in EP group presented weight loss compared with the mice in DD and PG groups after the 24th week. Seventy-nine macroscopic lesions were observed in the lingual mucosa, oral floor, and upper palatal and buccal mucosa. A total of 70 macroscopic lesions (88.6%) were located in the lingual mucosa. Mucosal lesions changed from simple hyperplasia to squamous cell carcinomas. Well-differentiated squamous cell carcinomas were observed in all mice of EP group by pathological section at the 28th week. No lesion was found in the mice of DD and PG groups.

CONCLUSIONThe animal model of lingual squamous cell carcinomas was successfully established. The periods from 12th to 16th week and 20th to 28th week were the ideal times for the research on pathogenesis of early and medial-advanced stage during carcinogenesis of squamous cell carcinomas.

4-Nitroquinoline-1-oxide ; Animals ; Cell Transformation, Neoplastic ; Disease Models, Animal ; Mice ; Mice, Inbred C57BL ; Mouth Mucosa ; Tongue

Objective To investigate the mechanism of morin-induced autophagy in non-small cell lung cancer A549 cells based on mTOR/STAT3 signaling axis.Methods A549 cells were divided into blank group and 30,60,90,120 and 150 μg·mL-1 of morin groups.After 24,48 and 72 hours of culture,the cell proliferation activity was detected by CCK-8 method,and the cell inhibition rate was calculated.A549 cells were divided into blank group and 30,90,150 μg·mL-1 morin groups.After 14 days of culture,the cell proliferation was detected by colony formation assay.After 24 hours of culture,the cell proliferation ability was detected by BeyoClickTM EdU-488.Apoptosis was detected by flow cytometry;acridine orange staining was used to detect cell autophagy;the formation of autophagosomes was observed by transmission electron microscopy.Western Blot was used to detect the expression levels of apoptosis,autophagy and mTOR/STAT3 signaling axis-related proteins in cells.A549 cells were divided into blank group,blank group + chloroquine(10 μg·mL-1)group,morin(30,150 μg·mL-1)group,morin(30,150 μg·mL-1)+ chloroquine(10 μg·mL-1)group.After 48 hours of intervention,the cell activity was detected by CCK-8 method,and the cell survival rate was calculated.Results Compared with the blank group,the inhibition rate of A549 cells in 60,90,120,150 μ g·mL-1 of morin group was significantly increased after 24 hours of intervention(P＜0.05,P＜0.001).The inhibition rates of A549 cells in 30,60,90,120 and 150 μg·mL-1 of morin groups were significantly increased after 48 and 72 hours of intervention(P＜0.001).The number of A549 cell colonies and the number of green fluorescent proliferation positive cells in the 30,90,150 μg·mL-1 of morin groups were significantly decreased(P＜0.01,P＜0.001),the apoptosis rate was significantly increased(P＜0.01,P＜0.001),and the protein expression level of cleaved-PARP was significantly increased(P＜0.001).The protein expression levels of p-P38/P38 MAPK in A549 cells of 90 and 150 μg·mL-1 of morin groups were significantly increased(P＜0.01,P＜0.001).Different degrees of orange fluorescence appeared in A549 cells of 30,90 and 150 μg·mL-1 of morin groups,and the orange fluorescence of 90 and 150 μg·mL-1 of morin groups was significant.Autophagosomes and autolysosomes appeared in the cytoplasm of A549 cells in 150 μg·mL-1 of morin group,respectively.The protein expression of LC3-Ⅱ in A549 cells of 150 μg·mL-1 of morin group was significantly up-regulated(P＜0.05).The protein expression of Atg16L1-Ⅱ in A549 cells of 90,150 μg·mL-1 of morin group was significantly up-regulated(P＜0.001),and the protein expressions of p-mTOR/mTOR and p-STAT3/STAT3 were significantly down-regulated(P＜0.001).Compared with the morin(150 μg·mL-1)group,the survival rate of A549 cells in the morin(150 μg·mL-1)+chloroquine(10 μg·mL-1)group was significantly increased(P＜0.05).Conclusion Morin can promote the apoptosis of A549 cells and induce autophagy in A549 cells,and the mechanism may be related to mTOR/STAT3 axis.

ObjectiveTo evaluate the effects of dehydrocostus lactone （DL） on the proliferation， apoptosis， and autophagy of human lung cancer cell A549 and to elucidate its related mechanism. MethodThe effect of DL with different concentrations （0， 5， 10， 15， 20， 25 μmol·L^-1） on the proliferation of human lung cancer A549 cells was investigated by cell counting kit-8 （CCK-8）， and its impact on the clonogenic ability of A549 cells was studied by cell clonogenic assay. The concentrations 10， 20 μmol·L^-1 were selected as DL low-dose group and high-dose group. Hoechst 33258 staining and western blot were used to observe the effect of DL on apoptosis of A549 cells. Autolysosomes were detected by acridine orange staining， and the expression level of microtubule-associated protein 1 light chain 3 （LC3） was determined by immunofluorescence and western blot. In addition， the effects of DL in combination with autophagy inhibitors bafilomycin A1 （BAF-A1） or 3-methyladenine （3-MA） on the autophagy of A549 cells was checked by CCK-8 assay. Finally， the role of DL in the regulation of A549 cell signaling pathway was explored by Western blot. ResultCompared with the conditions in the control group， the survival rate of A549 cells in the DL groups （10， 15， 20， 25 μmol·L^-1） was decreased （P<0.01）， and 5 μmol·L^-1 DL could inhibited the formation of A549 clone cells （P<0.01）， indicating that DL could inhibit the proliferation of human lung cancer A549 cells. The number of apoptotic cells was higher in both DL low-dose and high-dose groups than that in the control group， and the expression of apoptosis-related proteins poly （ADP ribose） polymerase （PARP） and B lymphocytoma-2 （Bcl-2）-associated X protein （Bax） were up-regulated （P<0.05， P<0.01）， while the expression of Bcl-2 was down-regulated （P<0.01） in DL high-dose group. The acridine orange staining showed that the orange fluorescence in the DL high-dose group was enhanced compared with that in the control group， indicating that DL could dramatically promote the formation of autolysosomes. Moreover， 20 μmol·L^-1 DL could increase the orange fluorescent particles of LC3 and up-regulated the expression level of LC3 Ⅱ （P<0.01）. After addition of autophagy inhibitors， the sensitivity of A549 cells to the effects of DL was attenuated （P<0.01）， which suggested that autophagy was involved in DL-induced A549 cell death. Compared with the control group， DL high-dose group had increased expression of autophagy-related protein 3 （Atg3） and autophagy-related protein 5 （Atg5） while reduced phosphorylation levels of protein kinase B （Akt）， mammalian target of rapamycin （mTOR） and signal transducer and activator of transcription 3 （STAT3） （P<0.05， P<0.01）. ConclusionDL could activate apoptosis and autophagy to inhibit the proliferation and clonogenic ability of A549 cells via suppressing Akt/mTOR/STAT3 signaling pathway.

Cancer still has elevated morbidity and mortality, which undoubtedly impacts the life quality of affected individuals. Remarkable advances have been made in cancer therapy, although the toxicities of traditional therapies remain an obvious challenge. Dahuang Zhechong Pill (DHZCP), developed by Zhongjing Zhang in the Synopsis of the Golden Chamber, represents an effective anticancer traditional Chinese medicine (TCM). In this review, it was found that DHZCP is therapeutically utilized in liver, lung, gastric, pancreatic and other cancers in clinic. Pharmacological evidence showed that its anti-tumor mechanisms mainly involve induced cell cycle arrest, apoptosis and autophagy, as well as suppressed tumor cell proliferation, obstructed angiogenesis and metastasis, enhanced immunity, and reversal of multidrug resistance. The present review provides a solid basis for the clinical application of DHZCP and may promote the wide use of TCM in clinical antitumor application.