| Background and objectivesColorectal cancer is one of the most common malignant tumors in the world,which seriously threatens human health and life safety.Clinically,colorectal cancer is difficult to find and easy to metastasize.Early colorectal cancer can usually be cured.However,many patients have developed to the advanced stage at the time of diagnosis accompanied by distant metastasis.At present,chemotherapy drugs for colorectal cancer such as 5-fluorouracil(5-FU)are mainly cytotoxic,which can not effectively inhibit the metastasis of advanced colorectal cancer,and always have severe side effects,which undoubtedly increases the difficulty of the treatment of colorectal cancer.Hypoxia is one of the key factors to promote colorectal cancer metastasis.By regulating oxidative metabolism,tumor cells can adapt to hypoxia microenvironment which induce angiogenesis and promote tumor invasion and metastasis.Therefore,the key to improve the curative effect of advanced colorectal cancer is to search for chemotherapeutics with low toxicity and good inhibitory effect on the metastasis under hypoxia.Quercetin is a natural compound derived from plants,and its anti-tumor activity has been widely reported in recent years.In vitro,quercetin has been reported to inhibit the proliferation,invasion and migration of tumor cells in colorectal cancer,breast cancer,prostate cancer,liver cancer and other cancers.However,the effect of quercetin on the invasion and metastasis of colorectal cancer,especially under hypoxia,and the effect of quercetin combined with 5-FU on the invasion and metastasis of advanced colorectal cancer under hypoxia have not been reported.In this paper,we constructed a hypoxia culture model in vitro,with high metastatic advanced colorectal cancer cell model as the research object and low metastatic colorectal cancer cell model in situ as the control,to explore whether quercetin can affect the proliferation,invasion,autophagy,migration and apoptosis of colorectal cancer under hypoxia and its potential molecular mechanism.Meanwhile,we observed the synergistic effect of quercetin and 5-FU on the invasion and metastasis of advanced colorectal cancer under hypoxia to explore the potential value of quercetin in the treatment of advanced colorectal cancer metastasis.Methods and resultsMethods1.Detect the toxic effect of quercetin on colorectal cancer cells under hypoxiaIn order to explore the toxic effect of quercetin on colorectal cancer cells under hypoxia,this paper selected the highly metastatic advanced colorectal cancer cell line LOVO and the low metastatic in situ colorectal cancer cell line HT-29 as the research objects.First,a hypoxia culture system was used to simulate the tumor hypoxia microenvironment(1%O2,5%CO2,37℃)to establish an in vitro hypoxia culture model.Then the CCK-8 method was used to detect the effects of different concentrations of quercetin on the survival rate of LOVO and HT-29 cells treated with quercetin for 24h,48h and 72h under hypoxia,and the IC50 was calculated.2.Detect the effect of quercetin on the invasion and migration ability of LOVO and HT-29 cells under hypoxiaIn order to explore the effect of quercetin on the invasion and migration of LOVO and HT-29 cells under hypoxia,Transwell assay and scratch test were used to detect the effect of quercetin on the invasion and migration of LOVO and HT-29 cells treated with 0,10,20 and 40 μmol/L quercetin under hypoxia for 24 hours.The expression levels of E-cadherin,N-cadherin,β-catenin,MMP-9,MMP-2 and Snail in LOVO cells treated with 0,10,20 and 40 μmol/L quercetin for 24 hours were detected by Western blot assay.In this experiment,a normoxia control group was designed.3.Detect the effect of quercetin on ROS and HIF-1α levels in LOVO cells under hypoxiaROS and HIF-1 a are usually highly expressed in tumor cells under hypoxia,which are the key factors to tumor hypoxia metabolism regulation.DCFH-DA fluorescent probe was uesd to label the ROS in LOVO cells treated with 0,10,20,40μmol/L quercetin for 24 hours,then use an inverted fluorescence microscope and flow cytometer to detect the green fluorescence intensity;Fluorescence method and inverted fluorescence microscope were used to detect the fluorescence intensity of HIF-1α in LOVO cells treated with 0,10,20,40 μmol/L quercetin for 24 hours.In this experiment,a normoxia control group was designed.4.Detect whether the effect of quercetin on the migration of LOVO cells under hypoxia depends on the PI3K/AKT signaling pathwayThe activation of PI3K/AKT is one of the key pathways to promote tumor cell invasion and migration.In order to explore whether the effect of quercetin on the migration of LOVO cells under hypoxia depends on the PI3K/AKT signaling pathway,the expression level of p-PI3K,PI3K,p-AKT,AKT protein in LOVO cells treated with 0,10,20,40 μmol/L quercetin for 24h under hypoxia were detected by Western blot assay.Then the AKT inhibitor MK-2206 was used to block the phosphorylation process of AKT under hypoxia,and the inhibitory effect of 40μmol/L quercetin treatment on the migration of LOVO cells for 24h under the premise of AKT inactivation was tested to verify whether the PI3K/AKT pathway is inhibited by quercetin.In this experiment,a normoxia control group was designed.5.Detect whether the effect of quercetin on the migration of LOVO cells under hypoxia depends on the Ras/MEK/ERK signaling pathwayThe activation of Ras/MEK/ERK pathway is one of the important mechanisms that promote tumor cell invasion and migration.PI3K/AKT signaling pathway and MEK/ERK signaling pathway are both downstream pathways of Ras.In order to explore whether the effect of quercetin on LOVO cell migration under hypoxia depends on the Ras/MEK/ERK signaling pathway,the expression levels of pan-Ras,p-MEK,MEK,p-ERK,ERK protein in LOVO cells treated with 0,10,20,40 μmol/L quercetin for 24h under hypoxia were detected by Western blot assay.In this experiment,a normoxia control group was designed.6.Detect the effect of quercetin on the autophagy of LOVO and HT-29 cells under hypoxiaIn order to detect the effect of quercetin on the autophagy of LOVO and HT-29 cells under hypoxia,an inverted fluorescence microscope was used to observe the morphology changes of LOVO and HT-29 cells treated with 0,20,40,80 μmol/L quercetin under hypoxia for 24h.Acridine orange staining method was used to observe the changes in the number of acidic autophagic vesicles in LOVO and HT-29 cells treated with 0,20,40,80 μmol/L quercetin under hypoxia for 24 hours.In the same time,Acridine orange staining method was used to observe the changes in the number of acidic autophagic vesicles in LOVO cells treated with 0,20,40,80 μmol/L quercetin under normoxia.7.Detect whether quercetin inhibits the migration of LOVO cells under hypoxia is related to its induction of autophagyIn order to determine whether quercetin inhibits the migration of LOVO cells under hypoxia is related to its induction of autophagy,bafilomycin A1,an autophagy inhibitor,was used to inhibit the autophagy of LOVO cells induced by quercetin under hypoxia.Inverted fluorescence microscope and acridine orange staining were used to observe the effect of combined use of 20 nmol/L bafilomycin on the autophagy of LOVO cells induced by 40 μmol/L under hypoxia.And t the effect of combined use of 20 nmol/L bafilomycin on the migration inhibition of LOVO cells induced by 40μmol/L under hypoxia were detected by scratch test.8.Detect the effect of quercetin on the apoptosis of LOVO and HT-29 cells under hypoxiaIn order to explore the effect of quercetin on the apoptosis of LOVO and HT-29 cells under hypoxia,Hoechst staining method and Annexin-FITC/PI double staining were used to qualitatively and quantitatively detect the apoptosis of LOVO and HT-29 cells treated with 0,20,40,80,160 μmol/L quercetin for 48h under hypoxia.In this experiment,a normoxia control group was designed.9.Detect the cytotoxic effect of quercetin and 5-FU on LOVO cells under hypoxiaIn order to explore the toxic effect of quercetin and 5-FU on LOVO cells under hypoxia,the CCK-8 method was used to detect the cytotoxic effect of combined treatment with 0,12.5,25,50 μmol/L 5-FU and 0,10,20,40 μmol/L quercetin on LOVO cells for 48h under hypoxia.10.Detect the effect of combination of quercetin and 5-FU on the invasion and migration ability of LOVO cells under hypoxiaIn order to explore the effect of quercetin and 5-FU on the invasion and migration of LOVO cells under hypoxia,the Transwell assay and scratch test were used to detect the effect of combined treatment with 25 μmol/L 5-FU and 40 μmol/L quercetin for 24h under hypoxia on the invasion and migration ability of LOVO cells for 24 hours.In this experiment,a normoxia control group was designed.Results1.Detect that quercetin inhibits the proliferation of LOVO and HT-29 cells under hypoxiaUnder hypoxia,quercetin significantly inhibited the proliferation of LOVO and HT-29 cells in a time-dependent and concentration-dependent manner.The IC50 of quercetin treatment on LOVO and HT-29 cells for 24h,48h and 72h were:347.43μmol/L,93.28μmol/L,59.65μmol/L;153.97μmol/L,85.18μmol/L,65.47μmol/L,respectively.Compared with the highly metastatic advanced colorectal cancer LOVO cell line,quercetin has a similar proliferation inhibitory effect on the low metastatic in situ colorectal cancer HT-29 cell line.2.Quercetin inhibits the invasion and migration of LOVO cells and the expression of related proteins under hypoxiaUnder hypoxia,quercetin significantly inhibited the invasion and migration of LOVO cells in a concentration-dependent manner.After treating LOVO cells with 10,20,and 40 μmol/L quercetin for 24 hours,the cell invasion rate was reduced to 82.12%,64.30%,26.35%,respectively,and the migration rate was reduced to 82.51%,40.14%,37.94%,respectively;The increased E-cadherin expression and decreased N-cadherin expression after quercetin treatment indicated that the EMT process is inhibited,and the expression levels of β-catenin,MMP-9,MMP-2,and Snail decrease,indicating that cell invasion and migration ability was inhibited.3.HT-29 has low invasion and migration potential under hypoxia and quercetin has no significant effect on thisCompared with the normoxic control,the number of HT-29 cells passing through the chamber was increased in the Transwell assay under hypoxia but the overall number was still small.At the same time,the scratch test showed that the migration rate of HT-29 cells did not change significantly after treatd with 0,10,20 and 40μmol/L quercetin for 24h under hypoxia with almost no migration occurred in each group.Experimental results shows that HT-29 has low invasion and migration potential under hypoxia and quercetin has no significant effect on this.4.Quercetin inhibited the expression of ROS and HIF-1α in LOVO cells under hypoxiaQuercetin significantly inhibited the expression of ROS and HIF-1α in LOVO cells in a concentration dependent manner.Compared with normoxic control group,the levels of ROS and HIF-1α in hypoxia control group were significantly increased.After treated with 10,20,40 μmol/L quercetin for 24 hours,the fluorescence intensity of ROS and HIF-1α in LOVO cells decreased significantly with the increase of Quercetin concentration,indicating that quercetin inhibited the expression of ROS and HIF-1α in LOVO cells under hypoxia.5.Quercetin inhibits the migration ability of LOVO cells by inhibiting the PI3K/AKT signaling pathway under hypoxiaAfter treatd with 10,20,and 40μmol/L quercetin for 24 hours under hypoxia,the expression levels of PI3K and p-AKT in LOVO cells was decreased and the AKT level does not change,indicating that quercetin inhibited PI3K/AKT signaling pathway in LOVO cells under hypoxia.After the combine use of MK-2206,the migration inhibitory effect of quercetin produced by AKT is masked,resulting in a weakened inhibitory effect on the migration of LOVO cells;Experimental results showed shows that quercetin inhibits the migration of LOVO cells by inhibiting the PI3K/AKT signaling pathway under hypoxia.6.Quercetin has no significant effect on the Ras/MEK/ERK signaling pathway in LOVO cells under hypoxiaAfter treating LOVO cells with 10,20,and 40 μmol/L quercetin for 24 hours,the expression levels of pan-Ras,p-MEK,MEK,p-ERK,and ERK did not change significantly,indicating that the effect of quercetin on LOVO cells under hypoxia has no close relationship with Ras/MEK/ERK signaling pathway.7.Quercetin induces autophagy in LOVO and HT-29 cells under hypoxiaAfter treating LOVO and HT-29 cells with 20,40,and 80 μmol/L quercetin for 24 hours under hypoxia,the boundaries of LOVO and HT-29 cells became unclear,the organelles shrunk aggravated,and vacuoles were seen in the cells Structure.Acridine orange staining showed that with the increase of quercetin concentration,the acidic autophagic vesicles stained red by acridine orange in LOVO and HT-29 cells increased significantly.Experimental results shows that quercetin induces autophagy in LOVO and HT-29 cells under hypoxia,which is stronger on the highly metastatic advanced colorectal cancer LOVO cell line than that of the low metastatic in situ colorectal cancer HT-29 cell line.8.Quercetin has no significant effect on the autophagy of LOVO cells under normoxic conditionsAfter treatment of LOVO cells with 20,40,and 80 μmol/L quercetin for 24 hours under normoxia,with the increase of quercetin concentration,the acidic autophagic vesicles stained red by acridine orange did not change significantly,indicating that quercetin has no significant effect on the autophagy of LOVO cells under normoxic conditions.9.Quercetin-induced autophagy and its inhibition of LOVO migration under hypoxia have no significant correlationThe autophagy level of the cells increased after treatment of LOVO cells with 40p,mol/L quercetin for 24h under hypoxia.After combined treatment with 20nmol/L Bafilomycin A1,the boundaries of LOVO cells became clear,the intracellular vacuole-like structure disappeared,and the acidic autophagic vesicles stained red by acridine orange in the cells were significantly reduced,indicating that 20nmol/L Bafilomycin A1 inhibited the autophagy of LOVO cells induced by quercetin under hypoxia.Then 20nmol/L Bafilomycin A1,40μmol/L quercetin and Bafilomycin A1 combined with quercetin were used to treat LOVO cells for 24h under hypoxia.Scratch test showed that Bafilomycin A1 combined with quercetin showed no statistically different migration rate compared with the quercetin alone group,indicating that the combined use of Bafilomycin A1 to inhibit quercetin-induced autophagy of LOVO cells has no significant effect on the inhibition of quercetin on the migration of LOVO cells under hypoxia.These results showed that under hypoxia,quercetin induced autophagy and its inhibition of LOVO migration has no obvious correlation.10.Quercetin induces LOVO and HT-29 cell apoptosis under hypoxiaAfter treating LOVO and HT-29 cells with 20,40,80,160 μmol/L quercetin for 24 hours,the kidney-shaped or horseshoe-shaped nucleus of cells appeared to be swelled and shattered or shrunk,and the chromatin was concentrated and deeply stained and showed strong blue fluorescence.Apoptotic bodies can be observed.With the increase of quercetin concentration,the number of apoptotic cells increases significantly.The results of flow cytometry showed that the apoptotic rates of LOVO cells treated with 20,40,80 and 160 μmol/L quercetin for 24 h and 48 h increased to 6.23%,30.67%and 12.33%,71.71%respectively.11.Quercetin had no significant effect on the inhibition of LOVO cell proliferation by 5-FU under hypoxiaUnder hypoxia,the IC50 of 5-FU combined with 0,10,20,and 40 μmol/L quercetin for 48 hours were 41.67μmol/L,39.40 μmol/L,47.23 μmol/L,36.82 μmol/L,respectively.After the combined use of different concentrations of quercetin,the inhibitory effect of 5-FU on the proliferation of LOVO cells did not change significantly,indicating that quercetin had no significant effect on the inhibition of LOVO cell proliferation by 5-FU under hypoxia.12.The combination of quercetin and 5-FU synergistically inhibits the invasion and migration of LOVO cells under hypoxia20μmol/L 5-FU and 40μmol/L quercetin were used alone or to treat LOVO cells for 24 hours under hypoxia.Compared with the hypoxia control group,the invasion and migration of LOVO cells were significantly inhibited,the invasion rate decreased from 65.25%and 38.36%to 28.93%,and the migration rate decreased from 69.66%and 31.21%to 24.75%.Conclusion1.Under hypoxia,quercetin inhibits the proliferation,invasion and migration of highly metastatic advanced colorectal cancer LOVO cells,induces LOVO cells autophagy and apoptosis;inhibits the proliferation of low metastatic in situ colorectal cancer HT-29 cells and induces HT-29 cells autophagy and apoptosis without affecting its invasion and migration ability.2.The inhibition of quercetin on the invasion and migration of LOVO cells under hypoxia may be related to its inhibition of ROS,HIF-1α expression levels and the PI3K/AKT pathway,but has no significant relationship with Ras/MEK/ERK signaling pathway and the autophagy of LOVO cells induced by quercetin。3.Under hypoxia,quercetin had no significant effect on the inhibition of LOVO cell proliferation by 5-FU under hypoxia.At the same time,the combination of quercetin and 5-FU can synergistically inhibit the invasion and migration of LOVO cells. |
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