| BackgroundIn recent years,with the continuous improvement of people’s living standards and the change of diet structure,the incidence rate and mortality of colorectal cancer in China are on the rise.A large number of clinical studies have confirmed that Traditional Chinese Medicine has its own unique advantages in prolonging the survival time of colon cancer patients,reducing the risk of recurrence,improving the quality of life and reducing the toxic and side effects of radiotherapy and chemotherapy.PiaoBingkui,the national famous Chinese medicine practitioner,specializes in the treatment of malignant tumors with Chinese and Western medicine,and has rich clinical experience.He believes that the main etiology of colon cancer is the weakness of the spleen and stomach,deficiency of qi and blood,he believes that supporting and regulating qi and detoxification is very important.The Fuzheng Tiaoqi Formula is a collection of Professor PiaoBingkui’s experience in the treatment of colon cancer,and our team has obtained this formula(Bai Zhu,Shan Yao,Zhi Qiao,and Yi Zhi Ren)by using the Liquorice system and complex network analysis technology.In this study,we investigated the mechanism of action of Fuzheng Tiaoqi Formula against colorectal cancer by using intestinal cancer cell lines(SW480,HCT116,HT29,CT26)and tumor-bearing mice,focusing on PI3K/AKT/mTOR signaling,to provide a basis and idea for subsequent research.ObjectivesTo investigate the possible mechanism of inhibiting intestinal cancer by regulating PI3K/AKT/mTOR signaling pathway through the Fuzheng Tiaoqi Formula,by observing the effects of the Fuzheng Tiaoqi Formula on proliferation,apoptosis,invasion and migration of intestinal cancer cell lines(SW480,HCT116,HT29,CT26)and tumor-bearing mice.MethodsIn vitro experiment1.Prepare serum containing Fuzheng Tiaoqi Formula,observe the effect of serum containing Fuzheng Tiaoqi Formula on the proliferation of colorectal cancer cell line,and determine the best concentration and time of administration;2.To study the effect of Fuzheng Tiaoqi Formula on the migration of colorectal cancer cell line by scratch experiment;3.To study the effect of Fuzheng Tiaoqi Formula on the invasion of colorectal cancer cell line by Transwell invasion test;4.To study the effects of serum containing Fuzheng Tiaoqi Formula,Fluorouracil,serum containing Fuzheng Tiaoqi Formula combined with Fluorouracil on the proliferation of intestinal cancer cells(SW480,HCT116,HT29 and CT26)by CCK8;5.To study the effects of serum containing Fuzheng Tiaoqi Formula and serum containing Fuzheng Tiaoqi Formula combined with Fluorouracil on the apoptosis rate of intestinal cancer cells(SW480,HCT116,HT29 and CT26)by flow cytometry AnnexinV-FITC/PI;6.To study the effects of expression of PI3K/AKT/mTOR signal pathway by Western Blot.In vivo experimentEstablish a model of CT26 bearing mice,intervene in the mice with Fluorouracil and Fuzheng Tiaoqi Formula combined with Fluorouracil,observe the volume and quality change of transplant tumor in each group of mice,and verify the inhibitory effect of Fuzheng Tiaoqi Formula combined with Fluorouracil on colon cancer in mice.Results1.Fuzheng Tiaoqi Formula can inhibit the proliferation of colon cancer cells SW480,HCT116,HT29 and CT26.When the concentration of drug-containing serum was 5%,the inhibition rate of Fuzheng Tiaoqi Formula on SW480 was(6.44±1.66)%in 24h,(7.78±1.62)%in 48h,and(4.88±1.57)%in 72h;When the concentration of drug-containing serum was 10%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(12.21±4.29)%,for 48 hours was(9.18±1.86)%,and for 72 hours was(7.13±3.46)%;When the concentration of drug-containing serum was 15%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(18.29±9.48)%,for 48 hours was(16.31±7.11)%,and for 72 hours was(9.90±2.23)%;When the concentration of drug-containing serum was 20%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(7.01±1.66)%,for 48 hours was(5.60±3.65)%,and for 72 hours was(7.56±3.27)%.When the concentration of medicated serum was 10%,the inhibition rate of Fuzheng Tiaoqi Formula on HCT116 was(6.38±3.47)%in 24h,(4.34±1,92)%in 48h,and(2.09 ± 1.96)%in 72h;When the concentration of drug containing serum was 15%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(8.33±1.33)%,for 48 hours was(15.94±1.87)%,and for 72 hours was(6.18±0.25)%;When the concentration of drug-containing serum was 20%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(19.07±3.17)%,for 48 hours was(19.07±4.16)%,and for 72 hours was(10.43±2.22)%;When the concentration of medicated serum was 25%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(1.66±0.74)%,for 48 hours was(5.35±2.75)%,and for 72 hours was(1.49±1.06)%.When the concentration of medicated serum was 5%,the inhibition rate of Fuzheng Tiaoqi Formula on HT29 was(5.09±4.01)%in 24h,(5.62±1.82)%in 48h,and(4.38±3.06)%in 72h;When the concentration of drug-containing serum was 10%,the inhibition rate of Fuzheng Tiaoqi Formula was(7.32±2.76)%in 24h,(1.78±5.52)%in 48h,(2.93±1.76)%in 72h;When the concentration of drug-containing serum was 15%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(13.72±2.77)%,for 48 hours was(8.25±1.34)%,and for 72 hours was(6.81±3.87)%;When the concentration of drug-containing serum was 20%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(12.21±3.22)%,for 48 hours was(2.04±4.01)%,and for 72 hours was(6.11±3.59)%.When the concentration of drug containing serum was 10%,the inhibition rate of Fuzheng Tiaoqi Formula on CT26 was(0.77±0.61)%in 24h,(5.13±5.08)%in 48h,(14.60±1.09)%in 72h;When the concentration of drug containing serum was 15%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(5.89±1.04)%,for 48 hours was(3.86±0.97)%,and for 72 hours was(11.51±1.77)%;When the concentration of drug containing serum was 20%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(15.43±2.92)%,for 48 hours was(14.56±5.24)%,and for 72 hours was(4.66±2.98)%;When the concentration of medicated serum was 25%,the inhibition rate of Fuzheng Tiaoqi Formula for 24 hours was(0.88±1.19)%,for 48 hours was(10.46±3.29)%,and for 72 hours was(10.35±5.70)%.Therefore,the optimal concentration of SW480 and HT29 was 15%,the optimal concentration of HCT116 and CT26 was 20%,and the optimal intervention time was 24h.2.Fuzheng Tiaoqi Formula can inhibit the migration of colon cancer cells S W480,HCT116,HT29 and CT26.The migration rate of SW480 in 24h was(21.60±1.79)%,which was significantly lower than that of(28.62±2.50)%in blank serum group and(35.33±2.63)%in model group(P<0.05).The migration rate of HCT116 in 24h was(17.15±3.39)%,which was significantly lower than that of(29.37±2.36)%in blank serum group and(30.91±1.80)%in model group(P<0.05).The migration rate of HT29 in 24h was(4.53±2.96)%,which was significantly lower than that of(11.17±2.08)%in blank serum group and(10.35±1.20)%in model group(P<0.05).The mean mobility of CT26 in 24h was(16.14 ± 4.39)%,which was significantly lower than that of(31.78±6.28)%in blank serum group and(43.31±5.45)%in model group(P<0.05).3.Fuzheng Tiaoqi Formula can inhibit the invasion of colorectal cancer cells.When the drug-containing serum of Fuzheng Tiaoqi Formula on SW480 in 24h,the number of membrane cells was(116.50±25.83),which was significantly lower than that of blank serum(272.83±51.45)(P<0.05).When the serum containing Fuzheng Tiaoqi Formula on HCT116 in 24h,the number of membrane cells was(123.67±6.11)significantly lower than that of blank serum(288.33±17.67)(P<0.05).When the serum containing Fuzheng Tiaoqi Formula on HT29 in 24h,the number of membrane cells was(113.66±14.50)less than that of blank serum(178.34±14.01)(P<0.05).When the serum containing Fuzheng Tiaoqi Formula on CT26 in 24h,the number of membrane cells was(189.34±18.56),which was significantly lower than that of blank serum(481.32±61.86)(P<0.05).4.Fuzheng Tiaoqi Formula combined with Fluorouracil can improve the inhibition rate of SW480,HCT116,HT29 and CT26.At the optimal concentration and time of drug-containing serum,the inhibition rate of Fuzheng Tiaoqi Formula combined with Fluorouracil(15mg/L)on SW480 was(45.36±6.52)%,which was better than(34.41±7.96)%in Fluorouracil group,and(27.63± 2.79)%in serum containing Fuzheng Tiaoqi Formula group;The inhibition rate of Fuzheng Tiaoqi Formula combined with Fluorouracil on HCT116 was(42.24±9.25)%,which was better than(30.85±8.56)%in Fluorouracil group and(19.07±5.26)%in serum containing Fuzheng Tiaoqi Formula group;The inhibition rate of Fuzheng Tiaoqi Formula combined with Fluorouracil on HT29 was(40.52±3.60)%,which was better than(27.85±4.27)%in Fluorouracil group,and(24.33±1.30)%in serum containing Fuzheng Tiaoqi Formula group;The inhibition rate of Fuzheng Tiaoqi Formula combined with Fluorouracil on CT26 was(48.60±1.66)%,which was better than(38.61±3.85)%in Fluorouracil group and(15.43±2.70)%in serum containing Fuzheng Tiaoqi Formula group.5.Fuzheng Tiaoqi Formula and its combined chemotherapy can increase the apoptosis rate of colorectal cancer cells SW480,HT29,HCT116 and CT26,and the effect of combined chemotherapy is more obvious.For SW480,the apoptosis rate of Fuzheng Tiaoqi Formula combined with 5FU group was(28.95±1.29)%,which was significantly higher than that of Fuzheng Tiaoqi Formula containing serum group(17.65±1.02)%,and that of 5FU group(21.26±2.26)%(P<0.05);The apoptosis rate of Fuzheng Tiaoqi Formula containing serum group on SW480 was(17.65±1.02)%,which was higher than that of blank serum group(13.97±1.32)%(P<0.05).For HCT116,the apoptosis rate of Fuzheng Tiaoqi Formula combined with 5FU group was(53.35±1.88)%,which was significantly higher than that of Fuzheng Tiaoqi Formula containing serum group(42.51±2.42)%,and that of 5FU group(43.33±4.74)%(P<0.05);The apoptosis rate of Fuzheng Tiaoqi Formula containing serum group on HCT116 was(42.51±2.42)%,which was higher than that of blank serum group(34.00±1.47)%(P<0.05).For HT29,the apoptosis rate of Fuzheng Tiaoqi Formula combined with 5FU group was(38.66±1.28)%,which was significantly higher than that of Fuzheng Tiaoqi Formula containing serum group(23.62±1.23)%,and that of 5FU group(29.73±0.86)%(P<0.05);The apoptosis rate of Fuzheng Tiaoqi Formula containing serum group on HT29 was(23.62±1.23)%,which was higher than that of blank serum group(14.68±1.35)%(P<0.05).For CT26,the apoptosis rate of Fuzheng Tiaoqi Formula combined with 5FU group was(36.49±2.93)%,which was significantly higher than that of Fuzheng Tiaoqi Formula containing serum group(24.47±1.78)%,and that of 5FU group(27.91± 3.07)%(P<0.05);The apoptosis rate of Fuzheng Tiaoqi Formula containing serum group on CT26 was(24.47±1.78)%,which was higher than that of blank serum group(18.03±4.64)%(P<0.05).6.Fuzheng Tiaoqi Formula combined with Fluorouracil inhibits the proliferation of colorectal cancer cells SW480,HCT116,HT29 and CT26,which is related to the regulation of PI3K/Akt/mTOR signal pathway.Fuzheng Tiaoqi Formula combined with Fluorouracil can inhibit the proliferation of SW480 by down regulating the protein expression of mTOR,p-mTOR,p-Akt and PI3K,and also changing the expression of ULK1 and p-ULK1;Fuzheng Tiaoqi Formula combined with Fluorouracil may inhibit the proliferation of HT29 by down regulating the protein expression of p-mTOR and p-Akt,and also changing the expression of ULK1 and LC3;Fuzheng Tiaoqi Formula combined with Fluorouracil may inhibit the proliferation of HCT116 by down regulating the protein expression of p-mTOR and p-Akt,and also changing the expression of ULK1;Fuzheng Tiaoqi Formula combined with Fluorouracil can inhibit the proliferation of CT26 by down regulating the protein expression of p-mTOR and p-Akt.At the same time,it also changes the expression of LC3 and p-ULK1.7.Fuzheng Tiaoqi Formula combined with Fluorouracil can inhibit the growth of tumor in CT26 tumor bearing mice.The inhibition rate(42.74%)is better than(24.36%)in 5FU group(P<0.05);The quality of life in the combined group was better than that in 5FU group,and the weight change was more stable.Fuzheng Tiaoqi Formula can improve the quality of life of tumor bearing mice and improve the curative effect of Fluorouracil.Conclusion1.Fuzheng Tiaoqi Formula inhibits the proliferation,invasion and migration of colorectal cancer cells;2.Fuzheng Tiaoqi Formula can induce apoptosis of intestinal cancer cells,and combined chemotherapy can improve its effect;3.Fuzheng Tiaoqi Formula combined with Fluorouracil may induce apoptosis of colorectal cancer cell line by regulating PI3K/AKT/mTOR signaling pathway;4.In vivo experiments further confirmed that Fuzheng Tiaoqi Formula combined with Fluorouracil can improve the anti-tumor effect. |
